Treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis with delayed-release 6-mercaptopurine

ABSTRACT

Methods of treating patients suffering from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), including those also suffering from type II diabetes mellitus (T2DM), with a delayed release pharmaceutical composition comprising 6-mercaptopurine are disclosed.

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/332,943, filed May 6, 2016 and 62/242,891, filedOct. 16, 2015, the entire contents of each of which are herebyincorporated by reference into the subject application.

Throughout this application, certain publications and patent applicationpublications are referenced. Full citations for the publications may befound immediately preceding the claims. The disclosures of thesepublications and patent application publications in their entireties arehereby incorporated by reference into this application in order todescribe more fully the state of the art to which this inventionrelates.

BACKGROUND OF THE INVENTION

Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) refers to a group ofconditions where there is accumulation of excess fat in the liver ofpeople who drink little or no alcohol. NAFLD comprises a wide spectrumof liver damage, ranging from simple macrovesicular steatosis tosteatohepatitis, advanced fibrosis, and cirrhosis (Sass et al. 2005).The most common form of NAFLD is a non-serious condition called fattyliver. In fatty liver, fat accumulates in the liver cells. Althoughhaving fat in the liver is not normal, by itself it probably does notdamage the liver (American College of Gastroenterology).

The majority of individuals with NAFLD have no symptoms and a normalexamination. Children may exhibit symptoms such as abdominal pain, whichmay be in the center or the right upper part of the abdomen, andsometimes fatigue. The liver might be slightly enlarged and somechildren may have patchy, dark discoloration of the skin present(acanthosis nigricans) most commonly over the neck and the under armarea (American College of Gastroenterology). Symptoms can also includeweight loss (Mayo Clinic).

Non-alcoholic fatty liver disease (NAFLD) is one of the causes of fattyliver, occurring when fat is deposited (steatosis) in the liver due tocauses other than excessive alcohol use. NAFLD is the most common liverdisorder in developed countries. NAFLD is related to insulin resistanceand the metabolic syndrome and is associated with diabetes mellitus type2 and hyperlipidemia and obesity.

NAFLD affects 30% of the world population and about 80% of obese people.

Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD,and is regarded as a major cause of cirrhosis of the liver of unknowncause and can lead to the development of end stage liver disease and toprimary liver cancer. About 2-8% of the world population suffers fromNASH.

Most patients with NAFLD will not develop NASH. NASH is a separateentity which is associated with unique genetic, metabolomic, lipidomic,and proteomic phenotypes all of which differentiate patients that sufferfrom NASH from those with NAFLD.

The only way to diagnose NASH is by a liver biopsy which differentiatepatients with NASH from those with NAFLD.

In many patients, nonalcoholic fatty liver disease (NAFLD) is associatedwith metabolic risk factors such as obesity, diabetes mellitus, anddyslipidemia. Nonalcoholic fatty liver disease is histologically furthercategorized into nonalcoholic fatty liver (NAFL) and nonalcoholoicsteatohepatitis (NASH).

Nonalcoholic fatty liver is defined as the presence of hepatic steatosiswith no evidence of hepatocellular injury in the form of ballooning ofthe hepatocytes. Nonalcoholic steatohepatitis is defined as the presenceof hepatic steatosis and inflammation with hepatocyte injury(ballooning) with or without fibrosis (Chalasani et al 2012).Nonalcoholic steatohepatitis can progress to cirrhosis, liver failure,and rarely liver cancer.

Non-alcoholic fatty liver disease, comprising non-alcoholic fatty liver(NAFL) and NASH, is a multi-system disease with hepatic and extrahepaticmanifestations, such as type 2 diabetes mellitus and cardiovasculardisease; it affects 30% of the adult population and at least 10% ofchildren (Loomba et al 2013, Anstee et al 2013). Non-alcoholicsteatohepatitis related liver transplantations are predicted to eclipseother indications over the next decade (Charlton et al 2011), and bothNAFLD and NASH have emerged as the dominant cause of hepatocellularcarcinoma (HCC), the only cancer with rising incidence and third leadingcause of cancer mortality (Friedman 2014).

Besides fatty liver, NAFLD also encompasses non-alcoholicsteatohepatitis and non-alcoholic fatty liver disease-associatedcirrhosis.

Non-Alcoholic Steatohepatitis

In a small number of people with fatty liver, the fat causesinflammation in the liver. This can impair the liver's ability tofunction and lead to scarring of the liver (cirrhosis). This is referredto as non-alcoholic steatohepatitis (NASH) (American College ofGastroenterology).

Liver inflammation leads to scarring of the liver tissue. With time,scarring can become so severe that the liver no longer functionsadequately (liver failure). This is known as non-alcoholic fatty liverdisease-associated cirrhosis (American College of Gastroenterology).

Treatment of the symptoms of NAFLD/NASH include vitamin E, lipidlowering medications, insulin sensitizing (medications), and thereduction of inflammation via anti-oxidant medications, anti-apoptoticmedications, and anti-cytokine medications. It may also includereduction of total cholesterol level, weight loss, control of anyunderlying diabetes, reduction or elimination of alcohol consumption,and regular exercise. (American College of Gastreoenterology; WebMD).However, there are currently no approved treatments for NASH itself.

Type II Diabetes Mellitus

Type II Diabetes Mellitus (T2DM) is a syndrome characterized byhyperglycemia resulting from absolute or relative impairment in insulinsecretion and/or insulin action. It is usually the type of diabetesdiagnosed in patients at least 30 years old, but also occurs in childrenand adolescents (Merck Manual, p. 165-177). Symptoms include polyuria(dilute urine), polydipsia (extreme thirst), polyphagia (extremehunger), weight loss, blurred vision, lower extremity paresthesias, oryeast infections, particularly balanitis in men (Medscape—Type IIDiabetes Mellitus). Genetic factors are the major determinants for T2DM(Merck Manual, p. 165-177).

Medicines used to treat T2DM include metformin (which helps the body useinsulin more effectively and lowers glucose production in the liver),sulfonylureas (which help the body secrete more insulin) such asglyburide, glipizide, and glimepiride, meglitinides (which alsoencourage the body to secrete more insulin), thiazolidinediones (whichmake the body's tissues more sensitive to insulin) such as rosiglitazoneand pioglitazone, DPP-4 inhibitors (which reduce blood sugar levels)such as sitagliptin, saxagliptin and linagliptin, GLP-1 receptoragonists (which slow digestion and help lower blood sugar levels), suchas exenatide and liraglutide, SGLT2 inhibitors (which prevent thekidneys from reabsorbing sugar in the blood), such as canagliflozin anddapagliflozin, and insulin therapy. Treatment for T2DM can also includea healthy diet and exercise (Medscape).

The presence of type 2 diabetes and other conditions associated withinsulin resistance, such as polycystic ovarian syndrome, are known riskfactors for the development of fatty liver and NASH (American College ofGastroenterology)

Obesity

Obesity is a chronic, relapsing health risk defined by excess body fat.The pathogenesis of obesity involves the interaction of genetic,environmental, and behavioral factors. Total body fat can be accuratelymeasured using hydrodensitometry and dual-energy x-ray absorptiometry(DEXA). Because body mass index (BMI), expressed as kilograms of weightdivided by height in meters squared (kg/m2), is simple and inexpensiveto calculate, and correlates strongly with total body fat in non-elderlyadults, it is commonly used as a surrogate for total body fat. Obesityis defined by the World Health Organization and the National Institutesof Health as a BMI of greater than or equal to 30. (U.S. Food and DrugAdministration, Guidance for Industry: Developing Products for WeightManagement, 2007)

6-Mercaotopurine

6-mercaptopurine (“6-MP”) is a synthetic analogue of natural purinebases (US Pub. No. 2006/0008520). After absorption into the body, it ispresumably transformed into nucleotides which interfere with nucleicacid biosynthesis, especially in the active S phase (US Pub. No.2006/0008520). As such, it is used to slow the growth of cancerous cells(US Pub. No. 2006/0008520).

6-MP received FDA approval for remission induction and maintenancetherapy of childhood acute lymphatic leukemia in 1953 (US Pub. No.2006/0008520). 6-MP is therefore indicated as a monotherapy and as partof combination therapies for treating acute lymphocytic leukemia in bothadults and children (Physician's Desk Reference 57th Edition, 2003, page1615-1618). A standard 6-MP (immediate release) 50 mg tablet isdescribed in Physician's Desk Reference 57th Edition, 2003, page1615-1618 and is sold in the United States under the brand namePURINETHOL®. 6-MP also exhibits immunosuppressive properties (US Pub.No. 2006/0008520).

U.S. Patent Application Publication Nos. 2006/0008520 and 2006/0009473,which are incorporated herein by reference in their entireties, disclosedelayed release pharmaceutical compositions comprising 6-MP (DR6-MP).

SUMMARY OF THE INVENTION

The invention provides for a method of treating a human patientsuffering from nonalcoholic fatty liver disease (NAFLD), obesity,metabolic syndrome, or any type of diabetes, including type 1 and type2, comprising periodically administering to the human patient a delayedrelease pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an amount of 6-mercaptopurine (6-MP) effective totreat the human patient.

The invention also provides for a method of treating a human patientsuffering from nonalcoholic steatohepatitis (NASH), comprisingperiodically administering to the human patient a delayed releasepharmaceutical composition comprising a pharmaceutically acceptablecarrier and an amount of 6-mercaptopurine (6-MP) effective to treat thehuman patient.

The invention also provides for the use of 6-mercaptopurine (6-MP) inthe manufacture of a medicament for the treatment of nonalcoholic fattyliver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for 6-mercaptopurine (6-MP) for use intreating a human patient afflicted with nonalcoholic fatty liver disease(NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for a pharmaceutical composition comprising6-mercaptopurine (6-MP) for use in the alleviation of a symptom, thetreatment, or the prevention, of nonalcoholic fatty liver disease(NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for a delayed release pharmaceuticalcomposition comprising 6-mercaptopurine, for the treatment, prevention,or alleviation of symptoms of nonalcoholic fatty liver disease (NAFLD)or nonalcoholic steatohepatitis (NASH).

The invention also provides for a package comprising

-   -   (a) a delayed release pharmaceutical composition comprising an        amount of 6-mercaptopurine (6-MP) and a pharmaceutically        acceptable carrier;    -   (b) instructions for use of the delayed release pharmaceutical        composition to treat a human patient suffering from nonalcoholic        fatty liver disease (NAFLD) or nonalcoholic steatohepatitis        (NASH).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Representative photomicrographs of Sirius red-stained liversections. Pathogen-free 14 day-pregnant C57BL/6 mice were obtained. NASHwas established in male mice by a single subcutaneous injection of 200τg streptozotocin after birth and feeding with a high fat diet adlibitum after 4 weeks of age (day 28±2). Mice were randomized into 5groups of 8 mice at 6 weeks of age (day 42±2), the day before the startof treatment. Group 1 (Normal): Eight normal mice were fed with normaldiet ad libitum without any treatment until 9 weeks of age. Group 2(Vehicle): Eight NASH mice were orally administered vehicle [0.5% Methylcellulose] in a volume of 10 mL/kg once daily from 6 to 9 weeks of age.Group 3 (Telmisartan): Eight NASH mice were orally administered purewater supplemented with Telmisartan at a dose of 10 mg/kg once dailyfrom 6 to 9 weeks of age. Group 4 (DR6-MP low dose): Eight NASH micewere orally administered vehicle supplemented with DR6-MP at a dose of10 mg/kg once daily from 6 to 9 weeks of age. Group 5 (DR6-MP highdose): Eight NASH mice were orally administered vehicle supplementedwith DR6-MP at a dose of 30 mg/kg once daily from 6 to 9 weeks of age.Mice were sacrificed at 6 to 9 weeks of age and liver sections werestained with Sirius-red.

FIG. 2: Fibrosis area. The Sirius-red stained liver sections obtained inthe protocol of FIG. 1 were used to estimate liver fibrosis area.Results were graphed and shown.

FIG. 3: Overall study schematic diagram. The figure represents theoverall study schematic.

FIG. 4: Serum ALT levels in treatment groups 1-3. NASH was induced inmice as in FIG. 1. Mice were randomized into 3 groups. Group 1(Vehicle): Eight NASH mice were orally administered vehicle [0.5% Methylcellulose] in a volume of 10 mL/kg once daily from 6 to 9 weeks of age.Group 2 (DR6-MP very low dose): Eight NASH mice were orally administeredvehicle supplemented with DR6-MP at a dose of 1 mg/kg once daily from 6to 9 weeks of age. Group 3 (Naïve+DR6-MP very low dose): Eight normalmice without receiving the streptozotocin injection were fed with normaldiet ad libitum and are orally administered vehicle supplemented withDR6-MP at a dose of 1 mg/kg once daily from 6 to 9 weeks of age. Micewere sacrificed at 9 weeks of age and serum ALT levels were measured andgraphed as shown.

FIG. 5: Fibrosis area of mice treated with very low dose DR6-MP.Pathogen-free 14 day-pregnant C57BL/6 mice were obtained. NASH wasestablished in male mice by a single subcutaneous injection of 200 μgstreptozotocin after birth and feeding with a high fat diet ad libitumafter 4 weeks of age (day 28±2). Mice were randomized into 2 groups of 8mice at 6 weeks of age (day 42±2), the day before the start oftreatment. Individual body weight was measured daily during thetreatment period. Survival, clinical signs and behavior of mice weremonitored daily. Food consumption was measured twice weekly per cageduring the treatment period. Group 1 (Vehicle): Eight NASH mice wereorally administered vehicle [0.5% Methyl cellulose] in a volume of 10mL/kg once daily from 6 to 9 weeks of age. Group 2 (DR6-MP very lowdose): Eight NASH mice were orally administered vehicle supplementedwith DR6-MP at a dose of 1 mg/kg once daily from 6 to 9 weeks of age.Group 3 (Naïve+DR6-MP very low dose): Eight normal mice withoutreceiving the streptozotocin injection were fed with normal diet adlibitum and are orally administered vehicle supplemented with DR6-MP ata dose of 1 mg/kg once daily from 6 to 9 weeks of age. Mice weresacrificed at 9 weeks of age and liver sections were stained withSirius-red.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for method of treating a human patient sufferingfrom nonalcoholic fatty liver disease (NAFLD), comprising periodicallyadministering to the human patient a delayed release pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and anamount of 6-mercaptopurine (6-MP) effective to treat the human patient.

In an embodiment of the invention, the NAFLD is simple steatosis.

In an embodiment of the invention, the NAFLD is nonalcoholicsteatohepatitis (NASH).

The invention also provides for a method of treating a human patientsuffering from NASH, comprising periodically administering to the humanpatient a delayed release pharmaceutical composition comprising apharmaceutically acceptable carrier and an amount of 6-MP effective totreat the human patient.

In an embodiment of the invention, the human patient is also sufferingfrom type II diabetes mellitus (T2DM).

In an embodiment of the invention, the human patient is also sufferingfrom type I diabetes mellitus (T1DM).

In an embodiment of the invention, the human patient is also sufferingfrom pre-diabetes or insulin resistance.

In an embodiment of the invention, the human patient is also sufferingfrom obesity, wherein obesity is defined as the patient having a bodymass index of ≥30.

In an embodiment of the invention, the patient has HbAlc levels between6.5-8.5%.

In an embodiment of the invention, the patient has a NAFLD activityscore of 4 or more.

In an embodiment of the invention, the patient has a NAFLD with anydegree of steatosis.

In an embodiment of the invention, the patient has evidence of liverfibrosis, inflammation or steatosis.

In an embodiment of the invention, the patient does not have evidence ofliver fibrosis.

In an embodiment of the invention, the patient has a score of at least 1in both steatosis and lobular inflammation.

In an embodiment of the invention, the patient has a hepatocyteballooning score of at least 1.

In an embodiment of the invention, the patient has a fibrosis score bytransient Elastography with Fibroscan of ≥F1.

In an embodiment, the patient is male and has an ALT level between 31and 105 IU/L, inclusive.

In an embodiment, the patient is female and has an ALT level between 20and 120 IU/L, inclusive.

In an embodiment, the patient has an MRI determined liver fat fractionof at least 10%.

In an embodiment of the invention, the patient is also beingadministered thiazolidinediones (glitazones), a stable dose for 3 monthsof dipeptidyl peptidase 4 inhibitors (gliptins), or glucagon-likepeptide-1 analogs or long acting insulin.

In an embodiment of the invention, the patient is also beingadministered metformin and/or sulfonylureas or DDP4 and/or long actinginsulin.

In an embodiment of the invention, the patient is also beingadministered immune modulatory agents.

In an embodiment of the invention, the immune modulatory agent issystemic steroids or daily treatment with non-steroidalanti-inflammatory drugs.

In an embodiment of the invention, the non-steroidal anti-inflammatorydrugs are aspirin in an amount of >100 mg/day, ibuprofen, naproxen,meloxicam and/or celecoxib.

In an embodiment of the invention, the patient is also beingadministered 5-ASA.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 0.0001 mg to 1000 mg of6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 1 mg to 120 mg of 6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 40 mg to 80 mg of 6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 40 mg of 6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 80 mg of 6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition administered to the patient contains 120 mg of 6-MP.

In an embodiment, the delayed release pharmaceutical compositionadministered to the patient contains 1-10 mg of 6-MP.

In an embodiment, the delayed release pharmaceutical compositionadministered to the patient contains 10-100 mg of 6-MP.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered daily.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered twice per day.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered less often than once daily.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered to a patient who is contemporaneouslyreceiving, or who has previously received, NAFLD or NASH therapy.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered to a patient who is contemporaneouslyreceiving any type of treatment for NAFLD. NASH, insulin resistance, ordiabetes.

In an embodiment of the invention, the NAFLD or NASH therapy is selectedfrom the group consisting of lipid-lowering medications,insulin-sensitizing medications, anti-oxidant medications,anti-apoptotic medications, and anti-cytokine medications.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered to a patient who is contemporaneouslyreceiving, or who has previously received. T2DM therapy.

In an embodiment of the invention, the T2DM therapy is selected from thegroup consisting of metformin, sulfonylureas, meglitinides,thiazolidinediones, DPP-4 inhibitors. GLP-1 receptor agonists, SGLT2inhibitors, and insulin therapy.

In an embodiment of the invention, the amount of 6-MP administered tothe patient per day is adjusted over time based on the patient'stolerability.

In an embodiment of the invention, the delayed release pharmaceuticalcomposition is administered for 24 weeks, and administration of thedelayed release pharmaceutical composition results in improvement of asymptom of NAFLD, NASH, or T2DM relative to baseline after 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 weeks of administration.

In an embodiment of the invention, the treatment results in theimprovement of a symptom of NAFLD or NASH, selected from the groupconsisting of weight loss, fatigue, enlarged liver, or skindiscoloration.

In an embodiment of the invention, the treatment results in theimprovement of a symptom of T2DM, selected from the group consisting ofpolyuria, polydipsia, polyphagia, weight loss, blurred vision, lowerextremity paresthesias, or a yeast infection.

In an embodiment of the invention, the treatment results in animprovement in insulin resistance.

In an embodiment of the invention, the treatment results in improvementof a symptom of pre diabetes.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in a reductionof HbAlc relative to baseline after 4, 12, or 24 weeks ofadministration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in HOMA or HOMA IR score relative to baseline after 24 weeksof administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in a reductionin percentage fat content of the liver relative to baseline after 24weeks of administration.

In an embodiment of the invention, the reduction in percentage fatcontent of the liver relative to baseline is ≥5%.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in anyreduction in steatosis.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in an ALTresponse after 24 weeks of administration, wherein the ALT response isan ALT level lower than 35 IU/L for women or an ALT level lower than 40IU/L for men.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in a reductionin serum ALT relative to baseline.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in a reductionin serum ALT relative to baseline after 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in no changeor a decrease in body weight relative to baseline after 2, 4, 12, or 24weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in serum lipid profile relative to baseline after 2, 4, 12,or 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in C-reactive protein (CRP) level relative to baseline after2, 4, 12, or 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in an immunological markers relative to baseline after 2, 4,12, or 24 weeks of administration.

In an embodiment of the invention, the immunological marker is selectedfrom the group consisting of CRP, erythrocyte sedimentation rate (ESR),interferon gamma (IFN-γ), CD4; CD8; CD4, CD25, FOXP3; CD3, CD56; CD4,CD62, and CD127.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient decreases the ESR.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient decreases CD62+expression.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in mean serum ALT relative to baseline after 2, 4, 8, 12,16, 20, or 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in mean serum aspartate aminotransferase (AST) relative tobaseline after 2, 4, 8, 12, 16, 20, or 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in GLP-1 level relative to baseline after 2, 4, 12, or 24weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in adiponectin level relative to baseline after 2, 4, 12, or24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in animprovement in fasting glucose or fasting insulin levels relative tobaseline after 2, 4, 12, or 24 weeks of administration.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in a reductionin liver fibrosis relative to baseline after 24 weeks of administration.

In an embodiment of the invention, the reduction in liver fibrosisrelative to baseline is assessed by elastography (Fibroscan) or by anynon invasive test for fibrosis or liver function.

In an embodiment of the invention, administration of the delayed releasepharmaceutical composition to the patient is effective to treat a liverdisease having liver fibrosis or inflammation as an underlying factor inthe liver disease or having liver fibrosis or inflammation associatedwith the pathogenesis of the liver disease.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective toprevent any liver disease having liver fibrosis or inflammation as anunderlying factor in the liver disease or having liver fibrosis orinflammation associated with the pathogenesis of the liver disease.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in theimprovement of a symptom of any liver disease having liver fibrosis orinflammation as an underlying factor in the liver disease or havingliver fibrosis or inflammation associated with the pathogenesis of theliver disease.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective to treatprimary sclerosing cholangitis (PSC).

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective toprevent PSC.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in theimprovement of a symptom of PSC.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective to treatprimary biliary cirrhosis (PBC).

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective toprevent PBC.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in theimprovement of a symptom of PBC.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective to treatautoimmune hepatitis (AIH) or graft versus host disease (GVHD).

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient is effective toprevent AIH or GVHD.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition to the patient results in theimprovement of a symptom of AIH or GVHD.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement in a bloodinflammation biomarker.

In an embodiment of the invention, the biomarker is TNF-α or FGF-19.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement in a liverfibrosis of cell death biomarker.

In an embodiment of the invention, the biomarker is CK-18 or sFas.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement in anoxidative stress biomarker.

In an embodiment of the invention, the biomarker ishydroxyeicosatetraenoic acids [HETEs], hydroxyoctadecadienoic acids[HODEs], oxoeicosatetraenoic acids [oxoETEs], or oxooctadecadienoicacids [oxoODEs].

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in resolution of NASH withoutworsening of fibrosis.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement on liverhistology in NASH subjects with fibrosis.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement inhistologic features of NASH from baseline to end of treatment liverbiopsy.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in an improvement in in NASHactivity, wherein NASH activity is defined by change in standardizedscoring of liver biopsies at baseline and at end of treatment.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in improvement in NASH by atleast 2 points spread across at least 2 of the NAS components.

In an embodiment of the invention, the administration of the delayedrelease pharmaceutical composition results in post treatment NAS of 3points or less.

In an embodiment of the invention, the patient is administered thedelayed release pharmaceutical composition for ≤24 weeks.

In an embodiment of the invention, the patient is administered thedelayed release pharmaceutical composition for ≥24 weeks.

The invention also provides for the use of 6-mercaptopurine (6-MP) inthe manufacture of a medicament for the treatment of nonalcoholic fattyliver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for 6-mercaptopurine (6-MP) for use intreating a human patient afflicted with nonalcoholic fatty liver disease(NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for a pharmaceutical composition comprising6-mercaptopurine (6-MP) for use in the alleviation of a symptom, thetreatment, or the prevention, of nonalcoholic fatty liver disease(NAFLD) or nonalcoholic steatohepatitis (NASH).

The invention also provides for a delayed release pharmaceuticalcomposition comprising 6-mercaptopurine, for the treatment, prevention,or alleviation of symptoms of nonalcoholic fatty liver disease (NAFLD)or nonalcoholic steatohepatitis (NASH).

The invention also provides for a package comprising

(a) a delayed release pharmaceutical composition comprising an amount of6-mercaptopurine (6-MP) and a pharmaceutically acceptable carrier;

(b) instructions for use of the delayed release pharmaceuticalcomposition to treat a human patient suffering from nonalcoholic fattyliver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

In an embodiment of the invention, the delayed release pharmaceuticalcomposition comprises 0.0001-1000 mg of 6-MP.

In an embodiment of the invention, the human patient is also sufferingfrom obesity, type II diabetes mellitus (T2DM), as well as type Idiabetes, and any type of insulin resistance

In an embodiment of the invention, the method includes a step ofdiagnosing the patient with NAFLD or NASH. Histologic diagnosis is basedon macrovesicular fatty change and lobular inflammation, sometimesaccompanies by fibrosis and Mallory hyaline bodies. The conditions mayalso be diagnosed by liver biopsies (Merck Manual, p. 366-368), or bymethods such as blood tests, abdominal ultrasounds, elastography, CTscan, or MRI scans (WebMD).

In an embodiment of the invention, the method includes a step ofdiagnosing the patient with T2DM. Diabetes is usually diagnosed viaglucose blood tests. Diagnostic criteria by the American DiabetesAssociation (ADA) include the following:

-   -   1. A fasting plasma glucose (FPG) level of 126 mg/dL (7.0        mmol/L) or higher, or    -   2. A 2-hour plasma glucose level of 200 mg/dL (11.1 mmol/L) or        higher during a 75-g oral glucose tolerance test (OGTT), or    -   3. A random plasma glucose of 200 mg/dL (11.1 mmol/L) or higher        in a patient with classic symptoms of hyperglycemia or        hyperglycemic crisis (Medscape).

HbAlc levels of 6.5% or higher are sometimes used as a diagnosticcriterion (Medscape).

Due to novel formulation and negligible systemic absorption compared to6MP, DR6-MP is believed to have an immunomodulatory effect rather thanimmunosuppression, with no effect on bone marrow suppression that couldinduce leucopenia.

For the foregoing embodiments, each embodiment disclosed herein iscontemplated as being applicable to each of the other disclosedembodiments.

All combinations, sub-combinations, and permutations of the variouselements of the methods described herein are envisaged and are withinthe scope of the invention.

Any delayed release 6-MP formulation may be for the purposes of theinvention, for example the DR6-MP formulations described in U.S. PatentApplication Publication No. US 2013/0280328 A1, the content of which ishereby incorporated by reference.

By any range disclosed herein, it is meant that all hundredth, tenth andinteger unit amounts within the range are specifically disclosed as partof the invention. Thus, for example, 10 mg to 60 mg means that 10.01,10.02 . . . 10.09; 10.1, 10.2 . . . 10.9: and 11, 12 . . . 59 mg unitamounts are included as embodiments of this invention. By any range oftime disclosed herein (i.e. weeks, months, or years), it is meant thatall lengths of time of days and/or weeks within the range arespecifically disclosed as part of the invention. Thus, for example, 3-6months means that 3 months and 1 day, 3 months and 1 week, and 4 monthsare included as embodiments of the invention.

Pharmaceutically Acceptable Salts

The active compounds for use according to the invention may be providedin any form suitable for the intended administration. Suitable formsinclude pharmaceutically (i.e. physiologically) acceptable salts, andpre- or prodrug forms of the compound of the invention.

Examples of pharmaceutically acceptable addition salts include, withoutlimitation, the non-toxic inorganic and organic acid addition salts suchas the hydrochloride, the hydrobromide, the nitrate, the perchlorate,the phosphate, the sulphate, the formate, the acetate, the aconate, theascorbate, the benzenesulphonate, the benzoate, the cinnamate, thecitrate, the embonate, the enantate, the fumarate, the glutamate, theglycolate, the lactate, the maleate, the malonate, the mandelate, themethane-sulphonate, the naphthalene-2-sulphonate, the phthalate, thesalicylate, the sorbate, the stearate, the succinate, the tartrate, thetoluene-p-sulphonate, and the like. Such salts may be formed byprocedures well known and described in the art.

Pharmaceutical Compositions

While the compounds for use according to the invention may beadministered in the form of the raw compound, it is preferred tointroduce the active ingredients, optionally in the form ofphysiologically acceptable salts, in a pharmaceutical compositiontogether with one or more adjuvants, excipients, carriers, buffers,diluents, and/or other customary pharmaceutical auxiliaries.

In an embodiment, the invention provides pharmaceutical compositionscomprising the active compounds or pharmaceutically acceptable salts orderivatives thereof, together with one or more pharmaceuticallyacceptable carriers therefore, and, optionally, other therapeutic and/orprophylactic ingredients know and used in the art. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not harmful to the recipient thereof.

The pharmaceutical composition of the invention may be administered byany convenient route, which suits the desired therapy. Preferred routesof administration include oral administration, in particular in tablet,in capsule, in dragée, in powder, or in liquid form, intranasaladministration, intradermal administration, and parenteraladministration, in particular cutaneous, subcutaneous, intramuscular, orintravenous injection. The pharmaceutical composition of the inventioncan be manufactured by the skilled person by use of standard methods andconventional techniques appropriate to the desired formulation. Whendesired, compositions adapted to give sustained release of the activeingredient may be employed.

The combination of the invention may be formulated for its simultaneous,separate or sequential administration, with at least a pharmaceuticallyacceptable carrier, additive, adjuvant or vehicle. This has theimplication that the combination of the two active compounds may beadministered:

-   -   as a combination that is being part of the same medicament        formulation, the two active compounds being then administered        always simultaneously.    -   as a combination of two units, each with one of the active        substances giving rise to the possibility of simultaneous,        sequential or separate administration.

Further details on techniques for formulation and administration may befound in the latest edition of Remintgton's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

The following abbreviations are used throughout this application:

5-ASA 5-aminosalicylic acid

6-MMP 6-methylmercaptopurine

6-MP 6-mercaptopurine

6-TG 6-thioguanine

6-TGN 6-thiohguanine nucleotide(s)

6-TUA 6-thiouric acid

6-TX 6-thioxanthine

8-OH-6-MP 8-hydroxy-6-mercaptopurine

ALP alkaline phosphatase

ALT alanine aminotransferase (SGPT)

ANC absolute neutrophil count

AST aspartate aminotransferase (SGOT)

AUClast area under the plasma drug concentration-time curve from time 0to the last measureable drug concentration

BMI body mass index

BP blood pressure

BUN blood urea nitrogen

CBC complete blood count

CD cluster of differentiation

CFR Code of Federal Regulations (US)

CI confidence interval

CK-18 cytokeratin-18

Cmax maximum observed drug concentration

CPK creatine phosphokinase

CPP clinical project physician

CRP C-reactive protein

DNA deoxyribonucleic acid

DR delayed release

DR6-MP delayed release 6-mercaptopurine

ECG electrocardiogram

ELISA enzyme-linked immunosorbent assay

ESR erythrocyte sedimentation rate

FACS fluorescence-activated cell sorting

FDA Food and Drug Administration (US)

FGF-19 fibroblast growth factor 19

FOXP3+ forkhead box P3

FSH follicle stimulating hormone

GGT Gamma-glutamyl transpeptidase

GI gastrointestinal

GLP-1 glucagon-like peptide-1

GVHD graft versus host disease

HbAlc glycosylated hemoglobin

HbsAg hepatitis B surface antigen

HCG human chorionic gonadotropin

HCV hepatitis C virus

HDL high-density lipoprotein

HETEs hydroxveicosatetraenoic acids

HIV human immunodeficiency virus

HODEs hydroxyoctadecadienoic acids

HOMA IR homeostatic model assessment of insulin resistance

hsCRP high-sensitivity C-reactive protein

HR heart rate

IB Investigator's Brochure

IEC Independent Ethics Committee

IFN interferon

IL interleukin

INN international nonproprietary name

IR immediate release

IRB Institutional Review Board

ITT intent to treat

K2EDTA dipotassium ethylenediaminetetraacetic acid

LC/MS/MS liquid chromatography/mass spectrometry/mass spectrometry

LDH lactate dehydrogenase

LDL low density lipoprotein

LFT liver function test

MedDRA Medical Dictionary for Regulatory Activities

min minimum

MRI Magnetic Resonance Imaging

MRI-PDFF magnetic resonance imaging-derived proton density-fat fraction

mRNA messenger ribonucleic acid

NA not applicable

NAFL nonalcoholic fatty liver

NAFLD nonalcoholic fatty liver disease

NAS NAFLD Activity Score

NASH nonalcoholic steatohepatitis

NIH National Institutes of Health (US)

OR odds ratio

ox-FA oxidized fatty acid profile

ox-NASH ox-nonalcoholic steatohepatitis

oxoETEs oxoeicosatetraenoic acids

oxoODEs oxooctadecadienoic acids

PBC primary biliary cirrhosis

PDAE protocol defined adverse event

PGx pharmacogenomics

PSC primary sclerosing cholangitis

qPCR quantitative polymerase chain reaction

Racc accumulation ratio

RBC red blood cell

RNA ribonucleic acid

RORγt retinoic acid-related orphan receptor-γt

rpm revolutions per minute

SAM-e S-adenosyl methionine

SD standard deviation

sFas soluble FAS

SOC system organ class

SOP standard operating procedure

TEAE treatment-emergent adverse events

T1DM Type 1 Diabetes Mellitus

T2DM Type 2 Diabetes Mellitus

TGF transforming growth factor

t_(max) time to maximum observed drug concentration

TNF tumor necrosis factor

TPMT thiopurine S-methyltransferase

US United States

V/F apparent total volume of distribution

WBC white blood cell

WHO World Health Organization

WHO Drug World Health Organization (WHO) drug dictionary

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

The articles “a”, “an” and “the” are non-limiting. For example. “themethod” includes the broadest definition of the meaning of the phrase,which can be more than one method.

As used herein, “about” in the context of a numerical value or rangemeans±10% of the numerical value or range recited or claimed.

As used herein, a subject or patient at “baseline” is as subject priorto administration of 6-MP in a therapy as described herein.

As used herein. “administering” to a subject means the giving of,dispensing of, or application of medicines, drugs, or remedies to asubject to relieve or cure a pathological condition. Oral administrationis an example of administration used in the instant methods.

As used herein, a “delayed release 6-MP pharmaceutical composition” or a“delayed release pharmaceutical composition comprising 6-MP” refers to apharmaceutical composition comprising 6-MP where release of 6-MP occursafter passage of the pharmaceutical composition through the stomach.Preferably, the pharmaceutical composition is enterically coated.Preferably, the enteric coating imparts a delay in the release in the6-MP following oral administration of the pharmaceutical compositionsuch that release of 6-MP occurs after passage of the compositionthrough the stomach. Optionally, the release of 6-MP occurs after atleast about 1 hour, at least about 2 hours, or at least about 3 hoursafter passage of the composition through the stomach. Alternatively, therelease of 6-MP occurs about 1 to about 3 hours or about 2 to about 3hours after passage of the composition through the stomach. Morepreferably, the release of 6-MP occurs about 5 to about 6 hours afteringestion.

As used herein. “effective” as in an amount effective to achieve an endmeans the quantity of a component that is sufficient to yield anindicated therapeutic response without undue adverse side effects (suchas toxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of thisdisclosure. The specific effective amount will vary with such factors asthe particular condition being treated, the physical condition of thepatient, the type of mammal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives.

As used herein, “periodic administration” means repeated/recurrentadministration separated by a period of time. The period of time betweenadministrations is preferably consistent from time to time. Periodicadministration can include administration, e.g., once daily, twicedaily, three times daily, four times daily, weekly, twice weekly, threetimes weekly, four times weekly and so on, etc.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or excipient that is suitable for use with humans and/or animalswithout undue adverse side effects (such as toxicity, irritation, andallergic response) commensurate with a reasonable benefit/risk ratio. Itcan be a pharmaceutically acceptable solvent, suspending agent orvehicle, for delivering compounds recited in the instant methods to thesubject.

As used herein “Adverse event” or “AE” means any untoward medicaloccurrence in a clinical trial subject administered a drug. An adverseevent can therefore be any unfavorable and unintended sign including anabnormal laboratory finding, symptom, or diseases temporally associatedwith the use of an investigational medicinal product, whether or notconsidered related to the investigational medicinal product. Adverseevents can be classified as Mild (no limitation of usual activities),Moderate (some limitation of usual activities) or Severe (inability tocarry out usual activities).

As used herein, to “treat” or “treating” encompasses, e.g., inducinginhibition, regression, or stasis of the disorder and/or disease. Asused herein. “inhibition” of disease progression or disease complicationin a subject means preventing or reducing the disease progression and/ordisease complication in the subject.

As used herein, “a patient afflicted with” or “a patient suffering from”NAFLD, NASH, or T2DM mean a subject who has been clinically diagnosed tohave NAFLD, NASH, or T2DM, respectively.

This invention will be better understood by reference to the Exampleswhich follow, but those skilled in the art will readily appreciate thatthe specific experiments are only illustrative of the invention asdescribed more fully in the claims which follow thereafter. Everyembodiment and feature described in the application should be understoodto be interchangeable and combinable with every embodiment containedwithin

Example 1: In Vivo Efficacy Study of DR6-MP in STAM Model ofNon-Alcoholic Steatohepatitis

Objective

To examine the effects of DR6-MP in the STAM model of non-alcoholicsteatohepatitis.

Methodology

Pathogen-free 14 day-pregnant C57BL/6 mice were obtained.

NASH was established in male mice by a single subcutaneous injection of200 μg streptozotocin after birth and feeding with a high fat diet adlibitum after 4 weeks of age (day 28±2).

Mice were randomized into 5 groups of 8 mice at 6 weeks of age (day42±2), the day before the start of treatment.

Individual body weight was measured daily during the treatment period.

Survival, clinical signs and behavior of mice were monitored daily.

Groups

Group 1 (Normal): Eight normal mice were fed with normal diet ad libitumwithout any treatment until 9 weeks of age.

Group 2 (Vehicle): Eight NASH mice were orally administered vehicle[0.5% Methyl cellulose] in a volume of 10 mL kg once daily from 6 to 9weeks of age.

Group 3 (Telmisartan): Eight NASH mice were orally administered purewater supplemented with Telmisartan at a dose of 10 mg/kg once dailyfrom 6 to 9 weeks of age.

Group 4 (DR6-MP low dose): Eight NASH mice were orally administeredvehicle supplemented with DR6-MP at a dose of 10 mg/kg once daily from 6to 9 weeks of age.

Group 5 (DR6-MP high dose): Eight NASH mice were orally administeredvehicle supplemented with DR6-MP at a dose of 30 mg/kg once daily from 6to 9 weeks of age.

Mice in all groups were sacrificed for the following assays at 9 weeksof age:

-   -   Measurement of organ weight        -   Individual liver weight was measured.        -   Liver-to-body weight ratio was calculated.    -   Biochemical assays        -   Serum ALT and AST levels were measured.        -   Liver triglyceride contents were quantified.    -   Histological analyses for liver sections (according to a routine        method).        -   HE staining and estimation of NAFLD Activity score.        -   Sirius-red staining and estimation of fibrosis area.        -   Immunochemical staining for F4/80 and estimation of the            percentage of inflammation area.    -   Gene expression assays using total RNA from the liver.        -   Real-time RT-PCR analyses were performed for TNF-α. INF-γ,            IL-10. MCP-1, CCR2, α-SMA, TGF-β, Collagen Type 1, Collagen            Type 3. TIMP-1.

Sample Collection

Samples of frozen serum and frozen liver were collected.

Statistical Tests

Statistical tests were performed using Bonferroni Multiple ComparisonTest. P values <0.05 were considered statistically significant.

Results

Representative photomicrographs of Sirius red-stained liver sections areshown in FIG. 1.

Liver sections from the Vehicle group showed increased collagendeposition in the pericentral region of liver lobule as compared withthe Normal group.

The Vehicle group showed a significant increase in fibrosis area (Siriusred-positive area) compared with the Normal group. The Telmisartan andDR6-MP low dose groups showed significant decreases in fibrosis areacompared with the Vehicle group.

TABLE 1 Histological analyses DR6-MP DR6-MP Parameter Normal VehicleTelmisartan low dose high dose (mean ± SD) (n = 8) (n = 8) (n = 8) (n =2) (n = 0) Sirius red- 0.23 ± 0.06 1.10 ± 0.18 0.74 ± 0.21 0.70 ± 0.13 —positive area (%)

Example 2: A 24-Week, Multicenter, Randomized, Double-Blind,Placebo-Controlled, Parallel-Group, Study to Evaluate the Efficacy andSafety of DR6-MP in Treating NASH in Patients with T2DM

Purpose and Objectives of the Study

The present study assesses if DR6-MP can improve liver health and liverfat content, as compared with placebo, in patients who suffer from T2DMand NASH.

The primary objectives of the study are to evaluate the effect of 6-MPtreatment on liver health, as compared with placebo treatment, byassessingserum alanine aminotransferase (ALT) levels and to evaluatewhether 6-MP treatment is more effective than placebo treatment inreducing liver fat content when measured by MRI-derived protondensity-fat fraction (MRI-PDFF). The comparison of serum ALT levels andliver fat content between 6-MP treatment and placebo treatment isconducted in adult patients with NASH and T2DM at week 24 (or the lastpostbaseline observation)

The secondary objectives of this study are to:

-   -   1) evaluate the effects of DR6-MP treatment compared with        placebo treatment on liver heath by assessing serum AST levels        after 24 weeks of treatment;    -   2) evaluate the effects of treatment on glycosylated hemoglobin        (HbAlc);    -   3) evaluate the effects of treatment on liver fibrosis, as        measured using transient Elastography with Fibroscan;    -   4) assess the pharmacokinetics of DR6-MP and its metabolites,        including 6-methylmercaptopurine (6-MMP), 6-thiouric acid        (6-TUA), and 6-thioguanine nucleotide(s) (6-TGN) in patients        with a historical diagnosis of NASH; and    -   5) evaluate the safety and tolerability of treatment as compared        with placebo treatment.

Exploratory objectives of this study are to:

-   -   1) determine the effect of DR6-MP on the immune profile based on        the following:        -   a. change from baseline in high-sensitivity C-reactive            protein (hsCRP) and erythrocyte sedimentation rate (ESR);        -   b. change from baseline in serum levels of tumor necrosis            factor alpha (TNF-α); transforming growth factor (TGF) beta;            interleukin (IL)-2, -4, -6, -10, and -12: and interferon            (IFN) gamma; and        -   c. fluorescence-activated cell sorting (FACS) analysis:            change from baseline in immunological markers (all patients)            (cluster of differentiation 3 [CD3], CD4, CD8, CD25, CD40,            CD56, CD69, CD127, forkhead box P3 [FOXP3+], IL17, and            retinoic acid-related orphan receptor-γt [RORγt]).    -   2) evaluate the effects of DR6-MP 80 mg once daily on blood        inflammation (TNF-α, fibroblast growth factor 19 [FGF-19]),        liver fibrosis of cell death biomarkers (cytokeratin-18 [CK-18],        soluable Fas [sFas]), and oxidative stress biomarkers—Oxidized        Fatty Acid Panel (hydroxyeicosatetraenoic acids [HETEs],        hydroxyoctadecadienoic acids [HODEs], oxoeicosatetraenoic acids        [oxoETEs], and oxooctadecadienoic acids [oxoODEs]) and        ox-nonalcoholic steatohepatitis (ox-NASH).    -   3) evaluate the effect of DR6-MP 80 mg once daily using the        homeostatic model assessment of insulin resistance (HOMA IR) to        measure insulin sensitivity.    -   4) evaluate the effect of DR6-MP 80 mg once daily, on serum        lipid profile (triglycerides, high-density lipoprotein [HDL],        low-density lipoprotein [LDL], and total cholesterol).    -   5) evaluate the effect of DR6-MP 80 mg once daily, on        glucagon-like peptide-1 (GLP-1) and adiponectin.    -   6) evaluate the relationship between        pharmacokinetic/pharmacodynamic relationship between DR6-MP and        metabolite concentrations and safety/pharmacodynamics endpoints.

HbAlc

HbAlc: a form of hemoglobin (also referred to as glycated hemoglobin)that is measured primarily to identify the average plasma glucoseconcentration over prolonged periods of time.

HOMA

The HOMA model is used to yield an estimate of j-cell function, insulinsensitivity, and insulin resistance (HOMA IR) from fasting plasmainsulin and glucose concentrations (Wallace et al. 2004; Matthews et al.1985; Levy et al. 1998).

Study Endpoints

Primary efficacy endpoints of this study are either at ALT response orat liver fat response. ALT response is defined as an ALT value withinthe reference ranges of <35 IU/L for women, or <40 IU/L for men. Liverfat response is defined as a reduction in liver fat of ≥6% as measuredby the MRI-PDFF. Both ALT response and liver fat response are assessedat week 24 of treatment.

Secondary efficacy endpoints include assessments of the change in ALTalone, in ALT and AST, in HbAlc. or the change in liver fibrosismeasured using transient elastography (with Fibroscan). The changes inthese parameters are all measured as compared to baseline. Theseendpoints may also be assessed at different time points.

Change in ALT alone is assessed in percent change from baseline at week24. Change in ALT and AST is measured as both percent change frombaseline, or numerical change from baseline, and is assessed at weeks 2,4, 8, 12, 16, 20, and 24 (or at early withdrawal of the patient from thestudy). Change in HbAlc is measured as numerical change from baseline,and is assessed at weeks 4, 12, and 24 (or at early withdrawal). Changein liver fibrosis is measured as numerical change from baseline, and isassessed at week 24 (or at early withdrawal).

Other secondary endpoints are an assessment of the plasma or whole bloodconcentrations and the pharmacokinetic parameters for DR6-MP and itsmetabolites 6-MMP, 6-TUA, and 6-TGN. These parameters are assessed atweeks 1, 2, 3, 4, 12, and 24 (or at early withdrawal), and at thefollow-up visit.

The exploratory endpoints of this study includes an evaluation of theeffect of DR6-MP on the immune profile, where the effect on the immuneprofile is characterized by the change from baseline in hsCRP and ESR atweeks 2, 4, 12, and 24 (or early withdrawal), change from baseline inserum levels of TNF-α, TGF beta, IL-2, IL-4, IL-6, IL-10, IL-12, and IFNgamma at weeks 2, 4, 12, and 24 (or early withdrawal), and FACSanalysis: change from baseline in immunological markers (CD3, CD4, CD8,CD25. CD40. CD56, CD69, CD127, FOXP3+, IL17, and RORγt) at 0 (predose)at weeks 2, 4, 12, and 24 (or early withdrawal).

The exploratory endpoints of this study also include:

-   -   1. the change from baseline on biomarkers of blood inflammation        (TNF-α, FGF-19), liver fibrosis of cell death biomarkers (CK-18,        sFas), and oxidative stress biomarkers Oxidized Fatty Acid Panel        (hydroxyeicosatetraenoic acids [HETEs], hydroxyoctadecadienoic        acids [HODEs], oxoeicosatetraenoic acids [oxoETEs], and        oxooctadecadienoic acids [oxoODEs])    -   2. the effects of DR6-MP treatment on ox-nonalcoholic        steatohepatitis [ox-NASH] at weeks 2, 4, 12, and 24 (or early        withdrawal);    -   3. change from baseline in fasting glucose and fasting insulin        at weeks 2, 4, 12, and 24 (or early withdrawal);    -   4. change from baseline in HOMA IR at weeks 2, 4, 12, and 24 (or        early withdrawal);    -   5. change from baseline in serum lipid profile at weeks 2, 4,        12, and 24 (or early withdrawal);    -   6. change from baseline in GLP-1 and adiponectin at weeks 2, 4,        12, and 24 (or early withdrawal);    -   7. percent change from baseline in body weight at weeks 2, 4,        12, and 24 (or early withdrawal);    -   8. pharmacokinetic/pharmacodynamic relationship between DR6-MP        and metabolite concentrations and safety/pharmacodynamic        endpoints;    -   9. the proportion of DR6-MP treated patients relative to placebo        achieving resolution of NASH without worsening of fibrosis;    -   10. evaluation of the effect of DR6-MP compared to placebo on        liver histology in NASH patients with fibrosis;    -   11. change in histologic features of NASH from baseline to end        of treatment liver biopsy;    -   12. number of participants with improvement in NASH activity, as        defined by change in standardized scoring of liver biopsies at        baseline and at the end of treatment;    -   13. improvement in NASH by at least 2 points spread across at        least 2 of the NAS components, or post treatment NAS of 3 points        or less.

In addition, pharmacogenomic (PGx) assessment includes assessment ofTPMT to determine eligibility for the study and might include otherdeoxyribonucleic acid (DNA) variations potentially associated withclinical treatment responses to DR6-MP (eg, clinical effect,pharmacokinetics, tolerability, and safety features or diseasesusceptibility and severity features).

Safety or tolerability endpoints are the evaluation of safety andtolerability of 24 weeks of treatment with DR6-MP on adverse events,physical examination findings, clinical laboratory evaluations (serumchemistry, hematology, and urinalysis) and 12-lead electrocardiograms(ECGs) from baseline to study completion, the early withdrawal of thepatient, including early withdrawal due to adverse events, or changes insafety laboratory test results (ALT, AST, total bilirubin, gammaglutamyltransferase [GGT], pancreatic amylase, uric acid and completeblood count (CBC) (and white blood cells [WBC] with differential andplatelet count). The safety laboratory test results are collected andmeasured at the following time points during the study: days −1 and 3and weeks 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or earlywithdrawal).

Methodology

A total of 80 T2DM and NASH subjects are randomized.

The patients are randomized into two groups:

-   -   One group receives a placebo, once daily (n=40)    -   One group receives a dose of DR6-MP, 80 mg, once daily (n=40)

The study consists of a screening (days −28 to −2), baseline (day −1), adouble-blind treatment (day 1 to week 24), and a follow-up/end of studyvisit.

DR6-MP is administered a dose of 80 mg per day. The dose may be titratedbased on patient tolerability, or it may be set at a fixed amount forthe duration of the study, regardless of tolerability.

DR6-MP may be administered in tablet form as described in U.S. PatentApplication Publication No. US 2013/0280328 A1, the content of which ishereby incorporated by reference, as shown in Table 1:

TABLE 1 Formulation of DR6-MP pharmaceutical composition (per tablet)Ingredient Weight (mg) Mercaptopurine 40 Microcrystalline Cellulose 280Citric Acid anhydrate 19.5 Potassium hydroxide 16.2 PVP K30 10.4Colloidal Silicon Dioxide 1.6 Potato Starch 24.4 Crospovidone 26.4Microcrystalline Cellulose 91.6 PVP K30 5.2 Magnesium Stearate 8.0Eudragit L100 77 Triethyl citrate 7.7 Talc 38.5

The study schematic diagram is presented in FIG. 3.

Elevated liver transaminases and increased liver fat content arehallmarks of steatohepatitis and thus changes in these markers are usedto assess the effect of treatment with DR6-MP.

Measures and Time Points:

Efficacy:

The primary efficacy measures and time points are as follows:

-   -   a) ALT response at week 24;        -   a. ALT value within the reference ranges of <35 IU/L for            women, or <40 IU/L for men    -   b) MRI-PDFF response at week 24.        -   a. a reduction in liver fat of ≥6% as measured by the            MRI-PDFF

The secondary efficacy measures and time points are as follows:

-   -   a) ALT and AST measurements at each visit after day 1 through        week 24 (or early withdrawal);    -   b) HbAlc measurements at baseline and weeks 4, 12, and 24 (or        early withdrawal);    -   c) Transient elastography (with Fibroscan) to assess liver        fibrosis at week 24 (or early withdrawal)

Safety:

The following safety and tolerability measures are implemented duringthe study:

-   -   a) Inquiries about adverse events at every visit;    -   b) Safety laboratory tests (ALT, AST, total bilirubin*, GGT,        pancreatic amylase, uric acid and CBC [and WBC with        differential] and platelet count) are collected and measured at        the following time points during the study: days 1 and 3, weeks        1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or early        withdrawal). *If total bilirubin values are above the normal        range then a direct and indirect bilirubin is determined;    -   c) Clinical laboratory (serum chemistry, hematology, and        urinalysis) tests at weeks 2, 4, 8, 12, and 24 (or early        withdrawal) and at the follow-up visit;    -   d) Vital signs (BP, pulse, respiratory rate) measured at every        visit;    -   e) An ECG recorded at baseline and week 24 (or early        withdrawal);    -   f) Physical examinations, including body weight measurements, at        baseline, week 24 (or early withdrawal), and at the follow-up        visit;    -   g) Inquiries about use of concomitant medication at every visit.

Exploratory/Other Measures and Time Points:

The exploratory measures and time points are as follows:

-   -   (a) plasma/whole blood concentrations of DR6-MP and its        metabolites: 6-MMP, 6-TUA, and 6-TGN predose at baseline, day 3,        and weeks 1, 2, 3, 4, and 24 (or earlywithdrawal), and at the        follow-up visit:    -   (b) whole blood ribonucleic acid (RNA) profile (including gene        expression and microribonucleic acid [mi-RNA]) and change from        baseline at weeks 2, 4, 12, and 24 (or early withdrawal);    -   (c) immune profile as characterized by the following:        -   a. hsCRP and ESR at baseline and weeks 2, 4, 12, and 24 (or            early withdrawal) and at the follow-up visit:        -   b. serum levels of inflammatory factors as TNF-α, TGF beta,            IL-2, IL-4, IL-6, IL-10, IL-12, and IFN gamma at baseline            and weeks 2, 4, 12, and 24 (or early withdrawal);        -   c. FACS analysis: immunological subpopulations including but            not limited to the following cell markers (CD3. CD4, CD8,            CD25, CD40, CD56, CD69, CD127, FOXP3+, IL17, and RORγt) at            baseline and weeks 2, 4, 12, and week 24 (or early            withdrawal);    -   (d) inflammation, fibrosis and cell death and oxidative stress        biomarkers at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal) (ie, TNF-α, FGF-19, CK-18, sFas, HETEs, HODEs,        oxoETEs, oxoODEs, and ox-NASH);    -   (e) fasting glucose and fasting insulin at baseline and weeks 2,        4, 12, and 24 (or early withdrawal);    -   (f) HOMA IR at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal):    -   (g) serum lipid profile at baseline and weeks 2, 4, 12, and 24        (or early withdrawal);    -   (h) GLP-1 and adiponectin at baseline and weeks 2, 4, 12, and 24        (or early withdrawal);    -   (i) body weight at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal);    -   (j) pharmacokinetic/pharmacodynamics relationship between DR6-MP        and metabolite concentrations and pharmacodynamic endpoints at        baseline, day 3, week 1, 2, 3, 4, 12, and 24.

Investigational Product:

Two DR6-MP tablets (40 mg) are self-administered orally once daily toprovide a dose of 80 mg.

Placebo:

Placebo is identical in appearance and packaging to the DR6-MP tablets.

Results

Primary Efficacy:

Treatment with 80 mg/day DR6-MP shows an ALT response after 24 weeks ofadministration, wherein the ALT response is defined as an ALT valuewithin the ranges of <35 IU/L for women, or <40 IU/L for men.

Treatment with 80 mg/day DR6-MP shows a decrease of 6% or more in liverfat at week 24 relative to baseline.

Secondary Efficacy:

Treatment with 80 mg/day DR6-MP shows a reduction in serum ALT levelsrelative to baseline after 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows reduction in serum levels of ALTand AST relative to baseline after 2, 4, 8, 12, 16, 20, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows a reduction in HbAlc relative tobaseline after 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows a reduction of liver fibrosisrelative to baseline after 24 weeks of administration.

Biomarkers:

Treatment with 80 mg/day DR6-MP shows an improvement in immune profilerelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of hsCRPand/or ESR relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum levels ofone or more of tumor necrosis factor alpha (TNF-α), transforming growthfactor (TGF) beta, interleukin (IL)-2, -4, -6, -10, and -12, and/orinterferon (IFN) gamma relative to baseline after 2, 4, 12, and/or 24weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of one ormore of CD3, CD4, CD8, CD25, CD40, CD56, CD69, CD127, FOXP3+, IL17,and/or RORγt relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of bloodinflammation, including TNF-α and/or fibroblast growth factor 19[FGF-19] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of liverfibrosis of cell death, including cytokeratin-18 [CK-18] and/or solubleFas [sFas] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers ofoxidative stress, including one or more of hydroxyeicosatetraenoic acids[HETEs], hydroxyoctadecadienoic acids [HODEs], oxoeicosatetraenoic acids[oxoETEs], and/or oxooctadecadienoic acids [oxoODEs] relative tobaseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in the levels ofox-nonalcoholic steatohepatitis biomarkers relative to baseline after 2,4, 12, and/or 24 weeks of administration.

Other Results:

Treatment with 80 mg/day DR6-MP shows an improvement in fasting glucoseand fasting insulin levels relative to baseline after 2, 4, 12, and/or24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in HOMA or HOMA IRscores relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum lipidprofile relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of GLP-1and/or adiponectin relative to baseline after 2, 4, 12, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows an improvement in body weightrelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows that a statistically significantproportion of treated patients achieve resolution of NASH withoutworsening of fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement on liver histologyin NASH subjects with fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement in histologicfeatures of NASH from baseline to end of treatment liver biopsy.

Treatment with 80 mg/day DR6-MP shows improvement in a statisticallysignificant number of patients over treatment with placebo in NASHactivity, wherein NASH activity is defined by change in standardizedscoring of liver biopsies at baseline and at end of treatment.

Treatment with 80 mg/day DR6-MP shows improvement in NASH by at least 2points spread across at least 2 of the NAS components.

Treatment with 80 mg/day DR6-MP shows post treatment NAS of 3 points orless.

Example 3: A 24-Week, Multicenter, Randomized, Double-Blind,Placebo-Controlled, Parallel-Group, Study to Evaluate the Efficacy andSafety of DR6-MP in Treating NASH in Patients with T1DM

A study is conducted according to the methods of Example 2, wherein theonly difference is that the patient inclusion criteria include T1DMinstead of T2DM, and the patient exclusion criteria include T2DM insteadof T1DM.

Results

Primary Efficacy:

Treatment with 80 mg/day DR6-MP shows an ALT response after 24 weeks ofadministration, wherein the ALT response is defined as an ALT valuewithin the ranges of <35 IU/L for women, or <40 IU/L for men.

Treatment with 80 mg/day DR6-MP shows a decrease of 6% or more in liverfat at week 24 relative to baseline.

Secondary Efficacy:

Treatment with 80 mg/day DR6-MP shows a reduction in serum ALT levelsrelative to baseline after 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows reduction in serum levels of ALTand AST relative to baseline after 2, 4, 8, 12, 16, 20, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows a reduction in HbAlc relative tobaseline after 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows a reduction of liver fibrosisrelative to baseline after 24 weeks of administration.

Biomarkers:

Treatment with 80 mg/day DR6-MP shows an improvement in immune profilerelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of hsCRPand/or ESR relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum levels ofone or more of tumor necrosis factor alpha (TNF-α), transforming growthfactor (TGF) beta, interleukin (IL)-2, -4, -6, -10, and -12, and/orinterferon (IFN) gamma relative to baseline after 2, 4, 12, and/or 24weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of one ormore of CD3, CD4, CD8, CD25, CD40. CD56, CD69, CD127, FOXP3+, IL17,and/or RORγt relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of bloodinflammation, including TNF-α and/or fibroblast growth factor 19[FGF-19] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of liverfibrosis of cell death, including cytokeratin-18 [CK-18] and/or solubleFas [sFas] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers ofoxidative stress, including one or more of hydroxyeicosatetraenoic acids[HETEs], hydroxyoctadecadienoic acids [HODEs], oxoeicosatetraenoic acids[oxoETEs], and/or oxooctadecadienoic acids [oxoODEs] relative tobaseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in the levels ofox-nonalcoholic steatohepatitis biomarkers relative to baseline after 2,4, 12, and/or 24 weeks of administration.

Other Results:

Treatment with 80 mg/day DR6-MP shows an improvement in fasting glucoseand fasting insulin levels relative to baseline after 2, 4, 12, and/or24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in HOMA or HOMA IRscores relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum lipidprofile relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of GLP-1and/or adiponectin relative to baseline after 2, 4, 12, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows an improvement in body weightrelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows that a statistically significantproportion of treated patients achieve resolution of NASH withoutworsening of fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement on liver histologyin NASH subjects with fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement in histologicfeatures of NASH from baseline to end of treatment liver biopsy.

Treatment with 80 mg/day DR6-MP shows improvement in a statisticallysignificant number of patients over treatment with placebo in NASHactivity, wherein NASH activity is defined by change in standardizedscoring of liver biopsies at baseline and at end of treatment.

Treatment with 80 mg/day DR6-MP shows improvement in NASH by at least 2points spread across at least 2 of the NAS components.

Treatment with 80 mg/day DR6-MP shows post treatment NAS of 3 points orless.

Example 4: A 24-Week, Multicenter, Randomized, Double-Blind,Placebo-Controlled, Parallel-Group, Study to Evaluate the Efficacy andSafety of DR6-MP in Treating NASH in Patients with Obesity

A study is conducted according to the methods of Example 2, wherein theonly difference is that the patient inclusion criteria include therequirement that the patients are obese, as defined as having a BMI of≥30 instead of T2DM.

Results

Primary Efficacy:

Treatment with 80 mg/day DR6-MP shows an ALT response after 24 weeks ofadministration, wherein the ALT response is defined as an ALT valuewithin the ranges of <35 IU/L for women, or <40 IU/L for men.

Treatment with 80 mg/day DR6-MP shows a decrease of 6% or more in liverfat at week 24 relative to baseline.

Secondary Efficacy:

Treatment with 80 mg/day DR6-MP shows a reduction in serum ALT levelsrelative to baseline after 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows reduction in serum levels of ALTand AST relative to baseline after 2, 4, 8, 12, 16, 20, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows a reduction in HbAlc relative tobaseline after 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows a reduction of liver fibrosisrelative to baseline after 24 weeks of administration.

Biomarkers:

Treatment with 80 mg/day DR6-MP shows an improvement in immune profilerelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of hsCRPand/or ESR relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum levels ofone or more of tumor necrosis factor alpha (TNF-α), transforming growthfactor (TGF) beta, interleukin (IL)-2, -4, -6, -10, and -12, and/orinterferon (IFN) gamma relative to baseline after 2, 4, 12, and/or 24weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of one ormore of CD3. CD4, CD8, CD25, CD40, CD56, CD69, CD127, FOXP3+, IL17,and/or RORγt relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of bloodinflammation, including TNF-α and/or fibroblast growth factor 19[FGF-19] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of liverfibrosis of cell death, including cytokeratin-18 [CK-18] and/or solubleFas [sFas] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers ofoxidative stress, including one or more of hydroxyeicosatetraenoic acids[HETEs], hydroxyoctadecadienoic acids [HODEs], oxoeicosatetraenoic acids[oxoETEs], and/or oxooctadecadienoic acids [oxoODEs] relative tobaseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in the levels ofox-nonalcoholic steatohepatliis biomarkers relative to baseline after 2,4, 12, and/or 24 weeks of administration.

Other Results:

Treatment with 80 mg/day DR6-MP shows an improvement in fasting glucoseand fasting insulin levels relative to baseline after 2, 4, 12, and/or24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in HOMA or HOMA IRscores relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum lipidprofile relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of GLP-1and/or adiponectin relative to baseline after 2, 4, 12, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows an improvement in body weightrelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows that a statistically significantproportion of treated patients achieve resolution of NASH withoutworsening of fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement on liver histologyin NASH subjects with fibrosis as compared to treatment with placebo.

Treatment with 80 mg/day DR6-MP shows an improvement in histologicfeatures of NASH from baseline to end of treatment liver biopsy.

Treatment with 80 mg/day DR6-MP shows improvement in a statisticallysignificant number of patients over treatment with placebo in NASHactivity, wherein NASH activity is defined by change in standardizedscoring of liver biopsies at baseline and at end of treatment.

Treatment with 80 mg/day DR6-MP shows improvement in NASH by at least 2points spread across at least 2 of the NAS components.

Treatment with 80 mg/day DR6-MP shows post treatment NAS of 3 points orless.

Example 5: A 24-Week, Randomized, Double-Blind, Placebo-Controlled,Study to Evaluate the Efficacy and Safety of DR6-MP in TreatingNon-Alcoholic Fatty Liver Disease in Patients Who Also have Type 2Diabetes Mellitus

Purpose and Objectives of the Study

The present study assesses if DR6-MP can improve liver fat content andliver health, as compared with placebo, in patients who suffer from bothT2DM and NAFLD.

The primary objectives of the study are to evaluate the effect of DR6-MPtreatment on liver fat content, as compared with placebo treatment, byassessing magnetic resonance imaging-derived proton density fat fraction(MRI-PDFF) after 24 weeks of treatment.

The secondary objectives of this study are to:

-   -   1) evaluate the effects of DR6-MP treatment, as compared with        placebo treatment, on liver health by assessing serum ALT levels        after 24 weeks of treatment:    -   2) evaluate the effects of DR6-MP treatment, as compared with        placebo treatment, on liver heath by assessing serum AST levels        after 24 weeks of treatment;    -   3) evaluate the effects of DR6-MP treatment on glycosylated        hemoglobin (HbAlc);    -   4) evaluate the effects of DR6-MP treatment on liver fibrosis,        as measured using transient Elastography with Fibroscan;    -   5) assess the pharmacokinetics of DR6-MP and its metabolites,        including 6-methylmercaptopurine (6-MMP), 6-thiouric acid        (6-TUA), and 6-thioguanine nucleotide(s) (6-TGN) in patients        with a historical diagnosis of NAFLD; and    -   6) evaluate the safety and tolerability of DR6-MP treatment as        compared with placebo treatment.

Exploratory objectives of this study are to:

-   -   1) determine the effect of DR6-MP on the immune profile based on        the following:        -   a. change from baseline in high-sensitivity C-reactive            protein (hsCRP) and erythrocyte sedimentation rate (ESR);        -   b. change from baseline in serum levels of tumor necrosis            factor alpha (TNF-α); transforming growth factor (TGF) beta:            interleukin (IL)-2, -4, -6, -10, and -12; and interferon            (IFN) gamma; and        -   c. fluorescence-activated cell sorting (FACS) analysis:            change from baseline in immunological markers (all patients)            (cluster of differentiation 3 [CD3], CD4, CD8, CD25. CD40,            CD56. CD69, CD127, forkhead box P3 [FOXP3+], IL17, and            retinoic acid-related orphan receptor-γt [RORγt]).    -   2) evaluate the effects of DR6-MP on blood inflammation (TNF-α,        fibroblast growth factor 19 [FGF-19]), liver fibrosis of cell        death biomarkers (cytokeratin-18 [CK-18], soluble Fas [sFas]),        and oxidative stress biomarkers Oxidized Fatty Acid Panel        (hydroxyeicosatetraenoic acids [HETEs], hydroxyoctadecadienoic        acids [HODEs], oxoeicosatetraenoic acids [oxoETEs], and        oxooctadecadienoic acids [oxoODEs]) and ox-nonalcoholic        steatohepatitis (ox-NASH).    -   3) evaluate the effect of DR6-MP using the homeostatic model        assessment of insulin resistance (HOMA IR) to measure insulin        sensitivity.    -   4) evaluate the effect of DR6-MP on serum lipid profile        (triglycerides, high-density lipoprotein [HDL], low-density        lipoprotein [LDL], and total cholesterol).    -   5) evaluate the effect of DR6-MP on glucagon-like peptide-1        (GLP-1) and adiponectin.    -   6) evaluate the relationship between        pharmacokinetic/pharmacodynamaic relationship between DR6-MP and        metabolite concentrations and safety/pharmacodynamics endpoints.

HbAlc

HbAlc: a form of hemoglobin (also referred to as glycated hemoglobin)that is measured primarily to identify the average plasma glucoseconcentration over prolonged periods of time.

HOMA

The HOMA model is used to yield an estimate of β-cell function, insulinsensitivity, and insulin resistance (HOMA IR) from fasting plasmainsulin and glucose concentrations (Wallace et al. 2004; Matthews et al.1985; Levy et al. 1998).

Study Endpoints

The primary efficacy endpoint are change from baseline in liver fat asassessed at week 24 by the MRI-PDFF and liver fat response.

Liver fat response is defined as a reduction in liver fat of ≥5% asmeasured by the MRI-PDFF.

Secondary efficacy endpoints include ALT response, which is defined as areduction in ALT serum levels from screening levels at week 24, as wellas assessments of the change in ALT alone, in ALT and AST, in HbAlc, orthe change in liver fibrosis measured using transient elastography (withFibroscan). The changes in these parameters (except for ALT response)are all measured as compared to baseline.

Change in ALT alone is assessed in percent change from baseline at week24. Change in ALT and AST is measured as both percent change frombaseline, or numerical change from baseline, and is assessed at weeks 2,4, 8, 12, 16, 20 and 24 (or at early withdrawal of the patient from thestudy). Change in HbAlc is measured as numerical change from baseline,and is assessed at weeks 4, 12, and 24 (or at early withdrawal). Changein liver fibrosis is measured as numerical change from baseline, and isassessed at week 24 (or at early withdrawal).

Other secondary endpoints are an assessment of the plasma or whole bloodconcentrations and the pharmacokinetic parameters for DR6-MP and itsmetabolites 6-MMP, 6-TUA, and 6-TGN. These parameters are assessed atweeks 1, 2, 3, 4, 12, and 24 (or at early withdrawal), and at thefollow-up visit.

The exploratory endpoints of this study also include the use of bloodsamples to conduct evaluations of:

-   -   1. whole blood ribonucleic acid (RNA) profile (including gene        expression and microribonucleic acid [mi-RNA]) and change from        baseline at weeks 2, 4, 12, and 24 (or early withdrawal)    -   2. immune profile as characterized by the following:        -   a. hsCRP and ESR at baseline and weeks 2, 4, 12, and 24 (or            early withdrawal) and at the follow-up visit        -   b. serum levels of inflammatory factors as TNF-α, TGF beta,            IL-2, IL-4, IL-6, IL-10, IL-12, and IFN gamma at baseline            and weeks 2, 4, 12, and 24 (or early withdrawal)        -   c. FACS analysis: immunological subpopulations including but            not limited to the following cell markers (CD3, CD4, CD8,            CD25, CD40, CD56, CD69, CD127, FOXP3+, IL17, and RORγt) at            baseline and weeks 2, 4, 12, and week 24 (or early            withdrawal)    -   3. inflammation, fibrosis and cell death and oxidative stress        biomarkers at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal) (i.e., TNF-α, FGF-19, CK-18, sFas, HETEs, HODEs,        oxoETEs, oxoODEs, and ox NASH)    -   4. fasting glucose and fasting insulin at baseline and weeks 2,        4, 12, and 24 (or early withdrawal)    -   5. HOMA IR at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal)    -   6. serum lipid profile at baseline and weeks 2, 4, 12, and 24        (or early withdrawal)    -   7. GLP- and adiponectin at baseline and weeks 2, 4, 12, and 24        (or early withdrawal)    -   8. percent change in body weight from baseline at weeks 2, 4,        12, and 24 (or early withdrawal)    -   9. pharmacokinetic/pharmacodynamic relationship between DR6-MP        and metabolite pharmacodynamics concentrations and endpoints at        baseline, day 3, week 1, 2, 3, 4, 12, and 24.

In addition, pharmacogenomic (PGx) assessment includes assessment ofTPMT to determine eligibility for the study and might include otherdeoxyribonucleic acid (DNA) variations potentially associated withclinical treatment responses to DR6-MP (e.g., clinical effect,pharmacokinetics, tolerability, and safety features or diseasesusceptibility and severity features).

Safety or tolerability endpoints include: inquiries about adverse eventsat every visit, safety laboratory tests (ALT, AST, total bilirubin*,GGT, pancreatic amylase, uric acid and CBC [and WBC with differential]and platelet count) are collected and measured (*If total bilirubinvalues are above the normal range then a direct and indirect bilirubinis determined), clinical laboratory (serum chemistry, hematology, andurinalysis) tests at weeks 2, 4, 8, 12, and 24 (or early withdrawal) andat the follow-up visit, vital signs (BP, pulse, respiratory rate)measured at every visit, an ECG recorded at baseline and week 24 (orearly withdrawal), physical examinations, including body weightmeasurements, at baseline, week 24 (or early withdrawal), and at thefollow-up visit, inquiries about use of concomitant medication at everyvisit. The safety laboratory test results are collected and measured atthe following time points during the study: days −1 and 3 and weeks 1,2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or early withdrawal)unless otherwise noted.

Methodology

A total of 80 T2DM and NAFLD subjects are randomized.

The patients are randomized into two groups:

-   -   One group receives a placebo, once daily (n=40)    -   One group receives a dose of DR6-MP, 80 mg, once daily (n=40)

The study consists of a screening (days −28 to −2), baseline (day −1), adouble-blind treatment (day 1 to week 24), and a follow-up/end of studyvisit.

DR6-MP is administered a dose of 80 mg per day. The dose may be titratedbased on patient tolerability, or it may be set at a fixed amount forthe duration of the study, regardless of tolerability. DR6-MP may beadministered in tablet form as described in U.S. Patent ApplicationPublication No. US 2013/0280328 A1, the content of which is herebyincorporated by reference, as shown in Table 1:

TABLE 3 Formulation of DR6-MP pharmaceutical composition (per tablet)Ingredient Weight (mg) Mercaptopurine 40 Microcrystalline Cellulose 280Citric Acid anhydrate 19.5 Potassium hydroxide 16.2 PVP K30 10.4Colloidal Silicon Dioxide 1.6 Potato Starch 24.4 Crospovidone 26.4Microcrystalline Cellulose 91.6 PVP K30 5.2 Magnesium Stearate 8.0Eudragit L100 77 Triethyl citrate 7.7 Talc 38.5

Screening

Patients are screened at visit 1 between days −28 and −2. At screening,patients undergo screening procedures meant to ensure thatinclusion/exclusion criteria are met, including an abdominal MRI toquantitatively measure liver fat content. Patients who meetinclusion/exclusion criteria based on the results of screeningassessments return to the study center on day −1 to undergo baselineassessments (visit 2). At the baseline visit, confirmation ofinclusion/exclusion criteria is done, and assessments of baselinelaboratory values, physical examination findings, and ECG results aredone.

Patients must have a certified histology report which documents andassesses the degree of steatosis, lobular inflammation, hepatocyteballooning, and fibrosis that confirms a diagnosis of NAFLD. A certifiedUS report showing nonalcoholic fatty liver disease and a transientElastography with Fibroscan assessment of liver fibrosis of ≥F1 can beused as alternatives for inclusion.

If patients are still eligible for participation following the baselinevisit, they return to the study center for the first day of study drugadministration on day 1 (visit 3). On day 1, patients are randomlyassigned to 1 of the 2 treatment groups (DR6-MP or placebo), receivetheir first dose of study medication at the study center under fastedconditions (patients are instructed to fast for 2 hours before takingthe study dose and through 2 hours after study drug administration eachday). Before they leave the clinic patients are instructed to take 2tablets with a glass of water every morning 2 hours before breakfastfrom each bottle.

Patients are also reminded to bring the study medication bottle(including any unused study medication) to the following study visit.Patients return to the site for visits 4 to 18 on day 3, day 7 (week 1),weeks 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or earlywithdrawal) days for safety and laboratory assessments (ALT, AST, totalbilirubin. GGT, pancreatic amylase, uric acid, and CBC (withdifferential) and platelet count). If clinically significant abnormalvalues are observed at any of the scheduled visits for laboratorymeasurements, the patient returns for a follow-up visit within 2 daysand additional laboratory assessments are completed. If increases inliver enzymes are observed, (based on criteria for discontinuation orwithdrawal of a patient) administration of the study drug isdiscontinued and the patient is followed until laboratory values aresimilar to those from the baseline visit.

At visit 18 on week 24 (or at early termination), all patients undergoend of treatment assessments, including liver fat content imaging by MRIand clinical laboratory safety assessments.

Liver imaging using transient elastography (measured by Fibroscan) iscompleted at screening and at week 24 (or at early termination).

Patients return 7 (±2) days after the last day on study drug (week 25)or 7 days (±2 days) after early termination for follow-up/end of study(visit 19). A final physical examination is conducted and blood iscollected for assessment of blood plasma concentration andpharmacokinetics. Vital signs, concomitant medications, and adverseevents are collected at every visit throughout the study.

Patients who complete all scheduled visits have procedures andassessments performed at the final visit. Patients who withdraw from thestudy before completing the 24-week evaluation period have earlytermination procedures and assessments (same procedures as those forvisit 18, week 24) performed at their final visit.

Elevated liver transaminases and increased liver fat content arehallmarks of steatohepatitis which is a disease resulting of damage tohepatic tissue. Measuring changes in laboratory markers and imaging ofhepatic tissue serve as appropriate measures and endpoints for thisexploratory proof of concept study to assess the effect of treatmentwith DR6-MP on patients with NAFLD who also have T2DM.

Patient Inclusion Criteria

Patients are included in this study only if they meet all of thefollowing criteria:

-   -   1. Age=18-65 years.    -   2. Gender—both    -   3. No healthy volunteers accepted    -   4. Provision of written informed consent.        -   a. capable of understanding and complying with protocol            requirements;        -   b. must be willing and able to comply with study            restrictions and to remain at the clinic for the required            duration during the study period, and willing to return to            the clinic for the follow-up evaluation as specified in this            protocol.    -   5. History of T2DM and on stable medication for diabetes or        insulin or a combination thereof for at least 3 months prior to        screening    -   6. The patient has T2DM with an HbAlc of 6.5 to ≤8.5 mg % at        screening.    -   7. Female patient:        -   a. has been surgically sterile for at least 24-weeks prior            to participation; or        -   b. is a postmenopausal woman who is amenorrhoeic for at            least 12 months (and has serum FSH level >30 IU/L at            Screening); or        -   c. if of child bearing potential, must be using a suitable            and effective contraceptive method and for 30 days after            last dose of study drug.            -   i. Must agree to not become pregnant during period of                study and for 30 days after the last dose of study drug.    -   8. Male patient:        -   a. is surgically sterile: or        -   b. if capable of producing offspring, is currently using an            approved method of birth control and agrees to continue use            of this method for the duration of the study (and for 30            days after taking the last does of study drug because of the            possible effects on spermatogenesis); or        -   c. has same-sex partners.        -   d. In addition to the above, male patients may not donate            sperm for the duration of the study and for 30 days after            taking the last dose of the study drug    -   9. If currently taking Vitamin E or pentoxifylline:        -   a. has been receiving a stable dose for 24 weeks prior to            randomization;        -   b. started Vitamin E therapy; and        -   c. agrees to maintain a stable dose throughout the study            when possible.    -   10. If currently taking a statin:        -   a. should be on stable does for 24 weeks prior to screening;            and        -   b. should agree to maintain that dose whenever possible            throughout the study.    -   11. Diagnosis of NASH, histologically proven within 12 months of        randomization based on historical histological evaluation or has        a certified US report showing NAFLD and a fibrosis score by        transient Elastography with Fibroscan of ≥F1.        -   a. no evidence of any other type of acute or chronic liver            disease    -   12. ALT 20-120 IU/mL, inclusive, for women, 31-105 IU/mL,        inclusive, for men at screening and at least once within        previous 24 weeks.    -   13. MRI determined liver fat fraction of at least 10% at        screening.

Patient Exclusion Criteria

Patients are excluded from participating in this study if they meet anyof the following criteria:

-   -   1. Current Conditions:        -   a. Type 1 diabetes or poorly controlled T2DM;        -   b. Any deficiency of the enzyme thiopurine methyl            transferase (via genotyping or other quantitative method).            If case assessment is done via genotyping, patients not            exhibiting TPMT*1/*1 genotype is excluded.        -   c. BMI ≤25 kg/m²;        -   d. Has any of the following clinically significant abnormal            laboratory values suggesting an undiagnosed disease other            than NASH requiring further clinical evaluation:            -   i. hemoglobin <12 g/dL for men and <10 g/dL for women;            -   ii. absolute neutrophil count (ANC) ≤1500/mm3;            -   iii. platelet count ≤150,000/mcL:            -   iv. WBC ≤3500 mcL;            -   v. serum albumin ≤3.5 g/dL;            -   vi. International Normalized Ratio (INR) ≥1.5;            -   vii. total bilirubin >1.5×upper limit of reference range                (unless Gilbert's syndrome or extrahepatic source as                denoted by increased indirect bilirubin fraction);            -   viii. either creatinine clearance ≤60 mL/minute                calculated by Cockroft Gault or creatinine >1.5× upper                limit of reference range.        -   e. Severe immunological diseases assessed at screening,            including:            -   i. HIV infection;            -   ii. Multiple sclerosis;            -   iii. Lupus erythematosus;            -   iv. Progressive multifocal leukoencephalopathy;        -   f. Any disorder which my interfere with drug absorption,            distribution, metabolism, or excretion (including GI            surgery);        -   g. Classified as Class II-IV via New York Heart Association:        -   h. Diastolic blood pressure greater than 100 mm Hg or a            systolic blood pressure greater than 160 mm Hg:            -   i. Based on mean of 3 serial BP measurements.        -   i. Has difficulty swallowing study medication        -   j. Seizures;        -   k. Currently participating in another clinical study;        -   l. Intolerance to venipuncture;        -   m. If female:            -   i. is pregnant, lactating, or intending to become                pregnant before, during, or within 1 month after                participating in this study; or                -   1. any woman becoming pregnant during the study is                    withdrawn from the study.            -   ii. intending to donate ova during, or within 1 month                after participating in this study:        -   n. Is an immediate family member, study site employee, or is            in a dependent relationship with a study site employee who            is involved in conduct of this study (e.g., spouse, parent,            child, sibling) or may consent under duress;        -   o. Has clinically significant results from physical            examinations or clinically significant laboratory results            that, at the discretion of the investigator, would make it            difficult to successfully manage and follow the patient            according to the protocol.    -   2. Medical History:        -   a. Chronic liver disease other than NASH (e.g., chronic or            acute hepatitis, autoimmune hepatitis, viral (A, B, C)            hepatitis, genetic hepatitis, drug induced hepatotoxicity,            Wilson's disease, alcoholic liver diseases, any other            non-NASH active liver disease);        -   b. Active cancer or history of a malignant disease (except            basal cell carcinoma of the skin) within 5 years prior to            screening or any history of bladder cancer;        -   c. Seizures;        -   d. Unstable metabolic condition (i.e. weight loss or gain of            5 kg or more in the past 24 weeks before screening);        -   e. Bariatric surgery within the last 5 years;        -   f. History of hypersensitivity, allergies, or any adverse            events related to 6-MP or azathioprine, including any            associated excipients;        -   g. Positive history of tuberculosis or a positive purified            protein derivative skin test which was not explained by            previous Bacillus Calmette-Guerin vaccination;        -   h. Diabetic gastroparesis or has had gastric bypass surgery            within the past 5 years:        -   i. Pancreatitis;        -   j. Persistent intestinal obstruction, bowel perforation,            uncontrolled GI bleed, abdominal abscess or infection, or            toxic megacolon or inflammatory bowel disease;        -   k. Coagulopathy;        -   l. Coronary angioplasty, coronary stent placement, coronary            bypass surgery, unstable angina, myocardial infraction,            transient ischemic events, or stroke within 24-weeks prior            to screening;        -   m. Illicit drug abuse;        -   n. Excessive alcohol abuse:            -   i. Men:                -   1. Regular or daily consumption of more than 3                    alcoholic drinks per day within 1 year prior to                    screening visit;            -   ii. Women:                -   1. Regular or daily consumption of more than 2                    alcoholic drinks per day within 1 year prior to                    screening visit;    -   3. Concurrent Medications or Medication History:        -   a. Has received an investigational medicinal product, or            participated in an investigational medical product research            study within a period of 3 months prior to the first dose of            the study drug;        -   b. Has received 6-MP in a previous clinical study or as a            therapeutic agent within 1 year prior to screening;        -   c. has been required to take excluded medications, including            anti-NASH therapy, for more than 1 continuous week in the            last 3 months. These include S-adenosyl methionine (SAM-c),            betaine, milk thistle, and probiotic supplements (other than            yogurt), but do not include vitamin E or gemfibrozil. The            following are exceptions to this exclusion criterion:            -   i. If vitamin E or gemfibrozil are used, the dose must                be stable subsequent to commencing study treatment;            -   ii. Thiazolidinediones (glitazones), stable dose for 3                months of dipeptidyl peptidase 4 inhibitors (gliptins)                or glucagon-like peptide-1 analogs or long acting                insulin in the last 24-weeks. Allowable anti-diabetic                treatment includes metformin and/or sulfonylureas or                DDP4 and/or long acting insulin, administered at                constant dose for at least 2 months prior to study                entry;            -   iii. Immune modulatory agents including, in the last 3                months, systemic steroids, daily treatment with                non-steroidal anti-inflammatory drugs (such as aspirin                (>100 mg/day), ibuprofen, naproxen, meloxicam,                celecoxib) within 4 weeks of study enrolment and/or                totaling 4 or more weeks in the last 3 months.        -   d. Has taken oral or injectable glucocorticoids for longer            than 7 consecutive days within the 3 months prior to            screening;        -   e. Has taken (or is scheduled to take during the trial)            anticholinergic or other drugs known to affect            gastrointestinal motility, or other drugs known to affect            gastric acidity or use of allopurinol, within 7 days prior            to the first dose of study drug;        -   f. Has received, within 24-weeks prior to first study dosing            (or is anticipated to take during the study),            immunosuppressants, including anti-TNF therapies (e.g.,            infliximab, adalimumab, etanercept), anti-integrin therapies            (e.g., vedolizumab, namixilab), oral antibiotics,            cyclosporine, tacrolimus, mycophenolate mofetil,            thalidomide, 6-MP, or azathioprine;        -   g. Has received oral antibiotics within the last 4 weeks            prior to randomization (day 1);        -   h. Has received treatment within the last 30 days with any            drugs known to induce or inhibit endogenous hepatic drug            metabolism, including:            -   i. Barbituates;            -   ii. Phenothiazines;            -   iii. Cimetidine;            -   iv. Carbamazepine;        -   i. Has taken anti-coagulant therapy within the last 30 days,            including:            -   i. Heparin;            -   ii. Warfarin;            -   iii. Acenocoumarol;

Patients are excluded who have other type of liver disease other thanNASH, hypersensitivity to 6-MP, uncontrolled diabetes, or anysignificant results from physical examinations or clinical laboratoryresults that would make it difficult to successfully manage and followthe patient according to the protocol.

Specifically, the inclusion and exclusion criteria were selected toensure that no patients are enrolled in the study if they have anydeficiency of the enzyme thiopurine methyl transferase or a condition inwhich they may have a history of allergy or hypersensitivity to DR6-MPor any of the metabolites. Patients who do not meet the strictconservative criteria based on clinical test results are excluded.

Biomarkers for evaluating safety (ALT, AST, total bilirubin. GGT,pancreatic amylase, uric acid, and CBC [and WBC with differential] andplatelet count) and efficacy are included and measured at baseline, andduring study conduct to be able to monitor patients safety and potentialbenefit in this patient population.

Criteria for Evaluation:

Safety:

Incidence and severity of adverse events and changes in vital signs andclinical laboratory tests.

Measures and Time Points:

Efficacy:

The primary efficacy measures and time points are as follows:

-   -   MRI-PDFF is used to evaluate the effect of DR6-MP 80-mg once        daily as compared to placebo, in reducing the liver fat content.        MRI-PDFF is performed at screening and week 24 or early        termination. Images are sent to a central imaging facility for        evaluation (details provided separately in imaging manual).

The secondary efficacy measures and time points are as follows:

-   -   a) Blood sampling for serum ALT levels to assess liver health is        performed at baseline, day 3, and at every subsequent visit.    -   b) ALT and AST measurements at each visit after day 1 through        week 24 (or early withdrawal);    -   c) HbAlc measurements at baseline and weeks 4, 12, and 24 (or        early withdrawal);    -   d) Transient elastography (with Fibroscan) to assess the effect        of DR6-MP on liver fibrosis at week 24 (or early withdrawal)

Safety:

The following safety and tolerability measures are implemented duringthe study:

-   -   a) Inquiries about adverse events at every visit;    -   b) Safety laboratory tests (ALT, AST, total bilirubin*, GGT,        pancreatic amylase, uric acid and CBC [and WBC with        differential] and platelet count) are collected and measured at        the following time points during the study: days −1 and 3, weeks        1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or early        withdrawal). *If total bilirubin values are above the normal        range then a direct and indirect bilirubin is determined;    -   c) Clinical laboratory (serum chemistry, hematology, and        urinalysis) tests at weeks 2, 4, 8, 12, and 24 (or early        withdrawal) and at the follow-up visit;    -   d) Vital signs (BP, pulse, respiratory rate) measured at every        visit;    -   e) An ECG recorded at baseline and week 24 (or early        withdrawal);    -   f) Physical examinations, including body weight measurements, at        baseline, week 24 (or early withdrawal), and at the follow-up        visit:    -   g) Inquiries about use of concomitant medication at every visit.

Exploratory/Other Measures and Time Points:

The exploratory measures and time points are as follows:

-   -   (a) plasma/whole blood concentrations of DR6-MP and its        metabolites: 6-MMP, 6-TUA, and 6-TGN predose at baseline, day 3,        and weeks 1, 2, 3, 4, and 24 (or early withdrawal), and at the        follow-up visit:    -   (b) whole blood ribonucleic acid (RNA) profile (including gene        expression and microribonucleic acid [mi-RNA]) and change from        baseline at weeks 2, 4, 12, and 24 (or early withdrawal);    -   (c) immune profile as characterized by the following:        -   a. hsCRP and ESR at baseline and weeks 2, 4, 12, and 24 (or            early withdrawal) and at the follow-up visit;        -   b. serum levels of inflammatory factors as TNF-α, TGF beta,            IL-2, IL-4. IL-6. IL-10, IL-12, and IFN gamma at baseline            and weeks 2, 4, 12, and 24 (or early withdrawal);        -   c. FACS analysis: immunological subpopulations including but            not limited to the following cell markers (CD3. CD4. CD8,            CD25. CD40, CD56. CD69, CD127, FOXP3+. IL17, and RORγt) at            baseline and weeks 2, 4, 12, and week 24 (or early            withdrawal);    -   (d) inflammation, fibrosis and cell death and oxidative stress        biomarkers at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal) (i.e., TNF-α, FGF-19, CK-18, sFas, HETEs, HODEs,        oxoETEs, oxoODEs, and ox-NASH);    -   (e) fasting glucose and fasting insulin at baseline and weeks 2,        4, 12, and 24 (or early withdrawal);    -   (f) HOMA IR at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal):    -   (g) serum lipid profile at baseline and weeks 2, 4, 12, and 24        (or early withdrawal):    -   (h) GLP-1 and adiponectin at baseline and weeks 2, 4, 12, and 24        (or early withdrawal);    -   (i) body weight at baseline and weeks 2, 4, 12, and 24 (or early        withdrawal);    -   (j) pharmacokinetic/pharmacodynamic relationship between DR6-MP        and metabolite concentrations and pharmacodynamics endpoints at        baseline, day 3, week 1, 2, 3, 4, 12, and 24.

Investigational Product:

Two DR6-MP tablets (40 mg) are self-administered orally once daily toprovide a dose of 80 mg.

Placebo:

Placebo is identical in appearance and packaging to the DR6-MP tablets.

Duration of Patient Participation and Justification

This study consists of a 28-day screening period, a 24-week double-blindtreatment period, and a 1-week follow-up period. Patients are expectedto participate in this study for its entire duration, which isapproximately 7 months (including screening and follow-up).

In order to observe an effect in the reduction and normalization of ALTand reduction of steatosis or liver fat, which are the primary endpointsselected for this study, literature and previous clinical studiessuggest that such changes should be observed as early as 3 months forALT and 4 to 6 months for measurements by MRI of the liver fat content.

Treatment of Patients

Restrictions

Throughout the double-blind treatment period, patients begin to fast atapproximately 2200 each evening and continue to fast for a minimum of 2hours after each study drug administration.

There is no restriction regarding consumption of water.

Prior and Concomitant Medication or Treatment

Any prior or concomitant medication, treatment, or procedure a patienthas had within 14 days before study drug administration and up to theend of study period, including follow-up, is recorded. Trade name orinternational nonproprietary name (INN), indication, and dose isrecorded.

Patients may be on a stable dose of metformin, dipeptidyl peptidase-4inhibitor, thiazolidinediones, sulfonylurea, or insulin or a combinationthereof; no changes in dosing regimen are allowed for 3 months prior toscreening and through the completion of all exit procedures, with theexception of those patients who meet the predefined Hyperglycemic RescueCriteria.

Patients must be on a stable (for at least 2 weeks prior to screening)dose of 5-aminosalicylic acid (5-ASA) and remain on the same dosethroughout the study duration. Immunosuppressive medications are to bewithdrawn 4 weeks prior to baseline, and not allowed during thetreatment period (e.g., cyclosporine, methotrexate, tumor necrosisfactor alpha receptor inhibitors or antibodies, gold, and azathioprine).

The following medications are not be allowed during this study:

-   -   anticholinergic or other drugs known to affect GI motility, or        other drugs known to affect gastric acidity or use of        allopurinol.    -   Note: H2 receptor antagonists are allowed until 8 days prior to        first study drug dose.    -   immunosuppressants such as anti-TNF therapies, cyclosporine,        tacrolimus, mycophenolate mofetil, thalidomide, or thioguanine    -   drugs known to inhibit endogenous hepatic drug metabolism such        as barbiturates, phenothiazines, cimetidine, carbamazepine, etc.    -   drugs known to cause myelosuppression    -   anticoagulation therapy such as heparin, warfarin, or        acenocoumarol    -   protease inhibitors including atazanavir, indinavir, nelfinavir,        ritonavir, and saquinavir, macrolide    -   antibiotics including erythromycin, clarithromycin, and        telithromycin    -   azole antifungals including itraconazole and ketoconazole,        nefazodone, fluoroquinolones including ciprofloxacin and        fluvoxamine, enoxacin, and cimetidine    -   Patients are instructed not to take any medications, including        over-the-counter products, without first consulting with the        Investigator.

Procedures for Monitoring Patient Compliance

A check of study drug compliance is performed during each visit afterthe initial dispensation of study drug; and study drug accountabilityrecords are completed. If the investigator or the sponsor determinesthat the patient is not in compliance with the study protocol, theinvestigator and the sponsor should determine whether the patient shouldbe withdrawn and the IEC/IRB should be notified.

Temporary Study Drug Discontinuation

Temporary study drug discontinuation is defined as missing more than 2consecutive days/doses of the study drug.

The patient must report any temporary study drug discontinuation to theinvestigator and is instructed by the investigator regardingcontinuation of treatment. The reasons for temporary study drugdiscontinuation are recorded and the medical monitor is notified.

Patients who discontinue study drug for 1 or 2 days may resume takingthe study drug at the same dose they were taking before the study drugdiscontinuation.

If a patient discontinues study drug for 3 or more days, study drugadministration may not be restarted.

Assessment of Efficacy

Primary Efficacy Measures and Justification

Several factors are possible contributors to NAFLD, such as insulinresistance, release of toxic inflammatory proteins by fat cells(cytokines), oxidative stress (deterioration of cells) inside livercells (characterized histopathologically by predominantly macrovesicularsteatosis with varying amounts of inflammation), cytological ballooning,and fibrosis. In order to observe an effect in the reduction andnormalization of ALT and reduction of steatosis or liver fat, literatureand previous clinical studies suggest that such changes could beobserved as early as 3 months for ALT and 4 to 6 months for measurementsby MRI of the liver fat content.

Blood sampling for serum ALT levels to assess liver health is performedat baseline, day 3, and at every subsequent visit except the follow-upvisit.

Magnetic resonance imaging-derived proton density-fat fraction is usedto evaluate the effect of DR6-MP 80-mg once daily as compared toplacebo, in reducing the liver fat content. Magnetic resonanceimaging-derived proton density-fat fraction is performed at baseline andweek 24 or early termination. Images are sent to a central imagingfacility for evaluation.

Assessment of Pharmacokinetics, Biomarkers, and Pharmacogenomics Studies

Pharmacokinetic Assessment

Plasma/whole blood samples are analyzed for concentrations of DR6-MP andits metabolites: 6-MMP, 6-TUA, and 6-TGN using an appropriate validatedmethod. Incurred sample reanalysis may be performed.

Specimen Sampling and Handling

Blood samples (6 mL) are collected via venipuncture for plasma/serumconcentration measurements of DR6-MP and metabolites or other analytes.

In the event of clinically significant abnormal laboratory valuesobserved at any time during the study, a blood sample is collected toevaluate plasma or whole blood concentrations for DR6-MP and itsmetabolites.

The dates and times of study drug administration and the date and timepoint of each pharmacokinetic sample are be recorded.

6-MP

Whole blood and plasma samples are kept in an ice-water bath during theentire sample collection and processing procedure.

6-MMP and 6-TGN

Whole blood samples are collected in pre-chilled labeled 2 mL bloodcollection tubes containing dipotassium ethylenediaminetetraacetic acid(K2EDTA) as the anticoagulant. The whole blood is divided into 2approximately equal aliquots. These whole blood tubes are stored in thefreezer at −80±15° C. within 45 minutes from the time of whole bloodcollection.

Alternatively, these samples are kept on dry ice until they aretransferred to a −80±15° C. freezer. The time at which samples areplaced into the freezer and the time they were stored on dry ice beforethat (if applicable) is recorded. During storage on dry ice, thetemperature is monitored if possible.

6-MP and 6-TUA

Whole blood samples are collected in pre-chilled labeled 4 mL bloodcollection tubes containing K2EDTA as the anticoagulant. The bloodsamples are then be centrifuged at approximately 4° C. for 10 minutes at3000 revolutions per minute (rpm). Samples that are interrupted duringcentrifugation, disturbed during the separation process or exhibitinadequate separation are re-spun under the same conditions in anattempt to separate the maximum amount of plasma from each sample. Thesamples are maintained in an ice-water bath following centrifugation.The centrifugation of whole blood is completed within 30 minutesfollowing blood draw. Separated plasma is transferred in approximatelyequal portions in 2 opaque, labeled, tubes (Sets A and B). These plasmatubes are stored in the freezer within 30 minutes from the time ofcentrifugation. Within 60 minutes of whole blood collection, the plasmaaliquots are stored at −80±15° C. pending assay. Alternatively, thesesamples are kept on dry ice until they are transferred to a −80±15° C.freezer. The time at which samples are placed into the freezer and thetime they were stored on dry ice before that (if applicable) isrecorded. During storage on dry ice, the temperature is monitored ifpossible.

Labels for samples include study number, patient randomization number,period, nominal collection time, Set A or B, and indication that theyare pharmacokinetic samples. Samples are stored at a temperature from−80±15° C. in an upright position until they are shipped to the centrallaboratory.

Pharmacogenomic Assessment

Pharmacogenomic assessment includes assessment of TPMT to determineeligibility for the study and might include other DNA variationspotentially associated with clinical treatment responses to DR6-MP(e.g., clinical effect, pharmacokinetics, tolerability, and safetyfeatures or disease susceptibility and severity features). Genomicanalysis could also include a sequencing of the whole genome ifrequired.

Thiopurine S-methyltransferase polymorphism is determined by a centrallaboratory for all patients, as part of the screening process todetermine eligibility for the study.

Results

Primary Efficacy:

Treatment with 80 mg/day DR6-MP shows a decrease in liver fat at week 24relative to baseline.

Secondary Efficacy:

Treatment with 80 mg/day DR6-MP shows a reduction in serum ALT levelsrelative to baseline after 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows reduction in serum levels of ALTand AST relative to baseline after 2, 4, 8, 12, 16, 20, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows a reduction in HbAlc relative tobaseline after 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows a reduction of liver fibrosisrelative to baseline after 24 weeks of administration.

Biomarkers:

Treatment with 80 mg/day DR6-MP shows an improvement in immune profilerelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of hsCRPand/or ESR relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum levels ofone or more of tumor necrosis factor alpha (TNF-α), transforming growthfactor (TGF) beta, interleukin (IL)-2, -4, -6, -10, and -12, and/orinterferon (IFN) gamma relative to baseline after 2, 4, 12, and/or 24weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of one ormore of CD3, CD4, CD8, CD25, CD40, CD56, CD69, CD127, FOXP3+, IL1117,and/or RORγt relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of bloodinflammation, including TNF-α and/or fibroblast growth factor 19[FGF-19] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers of liverfibrosis of cell death, including cytokeratin-18 [CK-18] and/or solubleFas [sFas] relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows a reduction in biomarkers ofoxidative stress, including one or more of hydroxyeicosatetraenoic acids[HETEs], hydroxyoctadecadienoic acids [HODEs], oxoeicosatetraenoic acids[oxoETEs], and/or oxooctadecadienoic acids [oxoODEs] relative tobaseline after 2, 4, 12, and/or 24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in the levels ofox-nonalcoholic steatohepatitis biomarkers relative to baseline after 2,4, 12, and/or 24 weeks of administration.

Other Results:

Treatment with 80 mg/day DR6-MP shows an improvement in fasting glucoseand fasting insulin levels relative to baseline after 2, 4, 12, and/or24 weeks of administration.

Treatment with 80 mg/day DR6-MP shows an improvement in HOMA or HOMA IRscores relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in serum lipidprofile relative to baseline after 2, 4, 12, and/or 24 weeks ofadministration.

Treatment with 80 mg/day DR6-MP shows an improvement in levels of GLP-1and/or adiponectin relative to baseline after 2, 4, 12, and/or 24 weeksof administration.

Treatment with 80 mg/day DR6-MP shows an improvement in body weightrelative to baseline after 2, 4, 12, and/or 24 weeks of administration.

Example 6: In Vivo Efficacy Study of DR6-MP at Very Low Dose in STAMModel of Non-Alcoholic Steatohepatitis

Objective

To examine the effects of DR6-MP in the STAM model of non-alcoholicsteatohepatitis.

Methodology

Pathogen-free 14 day-pregnant C57BL6 mice were obtained.

NASH was established in male mice by a single subcutaneous injection of2 μg streptozotocin after birth and feeding with a high fat diet adlibitum after 4 weeks of age (day 28±2).

Mice were randomized into 2 groups of 8 mice at 6 weeks of age (day42±2), the day before the start of treatment.

Individual body weight was measured daily during the treatment period.

Survival, clinical signs and behavior of mice were monitored daily.

Food consumption was measured twice weekly per cage during the treatmentperiod.

Groups

Group 1 (Vehicle): Eight NASH mice were orally administered vehicle[0.5% Methyl cellulose] in a volume of 10 mL/kg once daily from 6 to 9weeks of age.

Group 2 (DR6-MP very low dose): Eight NASH mice were orally administeredvehicle supplemented with DR6-MP at a dose of 1 mg/kg once daily from 6to 9 weeks of age.

Group 3 (Naïve+DR6-MP very low dose): Eight normal mice withoutreceiving the streptozotocin injection were fed with normal diet adlibitum and are orally administered vehicle supplemented with DR6-MP ata dose of 1 mg/kg once daily from 6 to 9 weeks of age.

Mice were euthanized for humane reasons if they show any of moribundity,severe weight loss (≥25% from study initiation or ≥20% within 24 hours)or decreasing spontaneous activity. All attempts are made to collectblood prior to the animal being placed in a chamber and euthanized underisoflurane anesthesia and prepare serum. The tissues are collected asdescribed below at study termination. If animals are found dead, thetissues are collected. At same time, a mouse in Group 3 (with the oldestID) is sacrificed and collected as described below at study termination.

Mice in all groups were sacrificed for the following assays at 9 weeksof age:

-   -   Measurement of organ weight        -   Individual liver weight was measured.        -   Liver-to-body weight ratio was calculated.    -   Biochemical assays        -   Serum ALT, AST, ALP and creatinine levels were measured.        -   Liver triglyceride contents are quantified.    -   Histological analyses for liver sections (according to a routine        method).        -   HE staining and estimation of NAFLD Activity score.        -   Sirius-red staining and estimation of fibrosis area.        -   Immunochemical staining for F4/80 and estimation of the            percentage of inflammation area.    -   Gene expression assays using total RNA from the liver.        -   Real-time RT-PCR analyses were performed for TNF-α, INF-γ,            IL-10, MCP-1, CCR2, α-SMA, TGF-β, Collagen Type 1, Collagen            Type 3, TIMP-1.    -   Preparation of lithium heparin plasma        -   Blood was collected in lithium heparin tube and centrifuged            as soon as possible (within 60 minutes after collection) at            1,000×g for 10 minutes at 4° C. The supernatant plasma was            separated then transferred into pre-labeled plasmatic tubes.            The plasma samples were stored at −80° C. after freezing            under liquid nitrogen. Tubes for plasma were labeled as            follows: study no, and animal number. From the sediment            (concentrated red blood cells. RBC), 2 aliquots (100 mg and            the rest) were prepared into prelabeled plastic tubes,            approx . . . 100 mg each and are stored at −80° (after            freezing under liquid nitrogen. Tubes for RBC sediment were            labeled as follows: study no., animal number, and RBC1 or            RBC2.

Whole blood was collected from animals prior to termination (24 to 27 hrafter the last dose) through direct cardiac puncture. Clear and precisedocumentation was provided about the time between sample collection andthe last dose administered.

Sample Collection

Samples of frozen lithium-heparin plasma, frozen liver, frozen RBCsediment, paraffin embedded liver block, paraffin embedded intestine andpancreas block, paraffin embedded spleen block, paraffin embedded lymphnode block, and bone marrow smear were collected.

Statistical Tests

Statistical tests are performed using Bonferroni Multiple ComparisonTest. P values <0.05 were considered statistically significant.

Results

Biochemical assays of the treatment groups showed that administration ofthe very low dose of DR6-MP (1 mg/kg) to NASH mice resulted in serum ALTlevels roughly equivalent to administration of vehicle alone.

FIG. 5 demonstrates that treatment of NASH mice with a very low dose ofDR6-MP (1 mg/kg) results in a significant decrease in fibrosis area inthe liver as measured by Sirius Red staining as compared to NASH micetreated with vehicle alone.

REFERENCES

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1. A method of treating a human patient suffering from nonalcoholicfatty liver disease (NAFLD), comprising periodically administering tothe human patient a delayed release pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an amount of6-mercaptopurine (6-MP) effective to treat the human patient.
 2. Themethod of claim 1, wherein the NAFLD is simple steatosis.
 3. (canceled)4. A method of treating a human patient suffering from nonalcoholicsteatohepatitis (NASH), comprising periodically administering to thehuman patient a delayed release pharmaceutical composition comprising apharmaceutically acceptable carrier and an amount of 6-MP effective totreat the human patient.
 5. The method of any of claim 1, wherein thehuman patient is also suffering from type II diabetes mellitus (T2DM),type I diabetes mellitus (T1DM), pre-diabetes or insulin resistance orobesity, wherein obesity is defined as the patient having a body massindex of ≤30. 6-9. (canceled)
 10. The method of claim 1, wherein thepatient has a NAFLD activity score of 0 or more.
 11. (canceled)
 12. Themethod of claim 1, wherein the patient has evidence of liver fibrosis,inflammation or steatosis.
 13. The method of claim 1, wherein thepatient does not have evidence of liver fibrosis. 14-15. (canceled) 16.The method of claim 12, wherein the patient has a fibrosis score bytransient Elastography with Fibroscan of ≤F1.
 17. The method of claim 1,wherein the patient is male and has an alanine aminotransferase (ALT)level between 31 and 105 IU/L, inclusive: or wherein the patient isfemale and has an ALT level between 20 and 120 IU/L, inclusive. 18-19.(canceled)
 20. The method of claim 1, wherein the patient is also beingadministered thiazolidinediones (glitazones), a stable dose for 3 monthsof dipeptidyl peptidase 4 inhibitors (gliptins), or glucagon-likepeptide-1 analogs or long acting insulin.
 21. The method of claim 1,wherein the patient is also being administered metformin and/orsulfonylureas or DDP4 and/or long acting insulin.
 22. The method ofclaim 1, wherein the patient is also being administered immunemodulatory agents. 23-24. (canceled)
 25. The method of claim 1, whereinthe patient is also being administered 5-ASA.
 26. The method of claim 1,wherein the delayed release pharmaceutical composition administered tothe patient contains 0.0001 mg to 1000 mg of 6-MP.
 27. The method ofclaim 26, wherein the delayed release pharmaceutical compositionadministered to the patient contains 1 mg to 120 mg of 6-MP. 28-33.(canceled)
 34. The method of claim 1, wherein the delayed releasepharmaceutical composition is administered daily or twice a day. 35-45.(canceled)
 46. The method of claim 1, wherein the treatment results inan improvement in insulin resistance. 47-95. (canceled)
 96. A method oftreating a human patient suffering from nonalcoholic fatty liver disease(NAFLD) or nonalcoholic steatohepatitis (NASH), comprising administeringto the human patient a therapeutically effective amount of6-mercaptopurine (6-MP). 97-100. (canceled)